BR112020005676A2 - new bispecific polypeptide complexes - Google Patents

Abstract

The present disclosure provides a polypeptide complex comprising variable regions of heavy chain and antibody light chain, respectively, fused to the regions contained in the TCR. Also provided here are a bispecific antigen-binding polypeptide complex containing a first antigen-binding portion of the polypeptide complex and a second antigen-binding portion, methods for producing the polypeptide complex or bispecific polypeptide-binding antigen complex, methods for the treatment of diseases or disorders with the use of the polypeptide complex or the bispecific polypeptide complex of antigen binding, polypeptides encoding the polypeptide complex and / or the bispecific polypeptide complex of antigen binding, vectors and host cells containing the polypeptides, pharmaceutical compositions and compositions comprising the polypeptide complex and / or the bispecific polypeptide complex binding to the antigen.

Description

“NEW BIESPECIFIC POLYPEPTIDE COMPLEXES” CROSS REFERENCE

[001] This application claims priority to International Patent Application No. PCT / CN2017 / 103.030, filed on September 22, 2017, the content of which is incorporated into this document in its entirety by reference.

FIELD OF THE INVENTION

[002] The present disclosure relates, in general, to soluble polypeptide complexes comprising antibody variable regions fused to TCR constant regions and bispecific polypeptide complexes comprising them.

FUNDAMENTALS

[003] Bispecific antibodies are becoming the new category of therapeutic antibodies. They can link two different targets or two different epitopes on a target, creating an additive or synergistic effect superior to that of individual antibodies. Many efforts to build antibodies have been dedicated to the development of new bispecific formats, such as DVD-Ig, CrossMab, BiTE etc. (Spiess et al., Molecular Immunology, 67 (2), pp. 95-106 (2015)). However, these formats can have several limitations regarding stability, solubility, short half-life and immunogenicity.

[004] Among these bispecific antibody formats, a bispecific IgG type antibody has a common format: one arm attaches to target A and another arm attaches to target B. Structurally, it is made from half of antibody A and half of antibody B, similar in size and shape to natural IgG.

In order to facilitate downstream development, it is desirable that such bispecific molecules can be easily produced as a normal IgG from a single host cell with a high level of expression and the correct assembled shape.

Unfortunately, the pairing of cognate light-heavy chains, as well as the assembly of two different half-antibodies, cannot be controlled automatically. All types of incorrect pairings at random can result in significant product heterogeneity.

[005] When introducing mutations in the Fc region, such as “knobs-into-holes” (Ridgway et al., Protein Engineering, 9 (7), pp.617-21 (1996); Merchant et al., Nature Biotechnology, 16 (7), pp. 67-681 (1998)), electrostatic (Gunasekaran et al., Journal of Biological Chemistry, 285 (25), pp.19637-19646 (2010)) or negative state projects (Kreudenstein et al. , mAbs, 5 (5), pp.646-654 (2013); Leaver-Fay et al., Structure, 24 (4), pp.641-651 (2016)), the preferred heterodimeric assembly of two different heavy chains it was made. However, the selective matching of the heavy-light chains of each individual antibody remains a challenge. The interface between light-heavy chains includes the variable domain (VH-VL) and the constant domain (CH1- CL). Several strategies have been applied in the development of orthogonal interfaces to facilitate cognate matching. Roche switched the CH1 and CL domains and created the CrossMab platform (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108 (27), pp.11187–11192 (2011)), As an alternative, MedImmune introduced the disulfide bond (Mazor et al., MAbs, 7 (2), pp. 37-389 (2015)), Amgen produced more electrostatics in the CH1-CL region (Liu et al., Journal of Biological Chemistry, 290 (12), pp.7535-7562 (2015)) and Lilly (Lewis et al., Nature Biotechnology, 32 (2), pp.191-198 (2014)) and Genentech (Dillon et al., mAbs, 9 (2), pp.213-230 (2017)) introduced mutations in the variable and constant domains.

[006] Therefore, there is a great need to develop bispecific molecules with desirable level of expression and affinity to antigens.

BRIEF SUMMARY OF THE INVENTION

[007] In one aspect, the present disclosure provides a polypeptide complex comprising a first polypeptide comprising, from the N-terminus to the C-terminus, a first heavy-chain variable domain

(VH) of a first antibody operably linked to a first constant region (C1) of the T cell receptor (TCR), and a second polypeptide comprising, from the N-terminal to the C-terminal, a first light chain variable domain ( VL) of the first antibody operably linked to a second constant region (C2) of the TCR, where C1 and C2 are capable of forming a dimer comprising at least one non-native interlink between C1 and C2, and the non-native interlinking is capable stabilize the dimer, and the first antibody has a first antigen specificity.

[008] In one aspect, the present disclosure provides a bispecific polypeptide complex comprising a first antigen binding portion associated with a second antigen binding portion, wherein the first antigen binding portion comprises a first polypeptide comprising, from the end N-terminal to C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the T cell receptor (TCR) and a second polypeptide comprising, from the N- terminal to C-terminal, a first variable light chain (VL) domain of the first antibody operably linked to a second constant region (C2) of the TCR, where C1 and C2 are capable of forming a dimer comprising at least one non-interlinked bond native between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, and the first antibody has a first antigen specificity, a second antigen binding portion has a second antigen specificity that is different from the first antigen specificity, and the first antigen binding portion and the second antigen binding portion are less prone to incorrect pairing of what it would be otherwise if both the first and second antigen-binding portions were equivalent to natural Fab.

[009] In one aspect, the present disclosure here provides a bispecific polypeptide complex comprising a first antigen binding portion comprising the polypeptide complex provided here with a first antigen specificity, associated with a second antigen binding portion with a second specificity antigen that is different from the first antigen specificity, and the first antigen-binding portion and the second antigen-binding portion are less prone to mismatch than it would otherwise be if both the first and second antigen-binding portions were equivalent to natural Fab.

[010] In one aspect, the present disclosure provides a bispecific fragment of the bispecific polypeptide complex provided here.

[011] In one aspect, the present disclosure here provides a conjugate comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided here conjugated to a moiety.

[012] In one aspect, the present disclosure here provides an isolated polynucleotide that encodes the polypeptide complex provided here, or the bispecific polypeptide complex provided here.

[013] In one aspect, the present disclosure here provides an isolated vector comprising the polynucleotide provided here.

[014] In one aspect, the present disclosure here provides a host cell comprising the isolated polynucleotide provided here or the isolated vector provided here.

[015] In one aspect, the present disclosure here provides a method for expressing the polypeptide complex provided here, or the bispecific polypeptide complex provided here, comprising cultivating the host cell provided here under the condition in which the polypeptide complex or the bispecific polypeptide complex is expressed.

[016] In one aspect, the present disclosure here provides a method for producing the polypeptide complex provided herein, comprising a) introducing into a host cell a first polynucleotide encoding a first polypeptide comprising, from the N-terminal to the C-terminal end, a first heavy chain variable domain (VH) of a first antibody operably linked to a first constant domain (C1) of the TCR, and a second polynucleotide encoding a second polypeptide comprising, from the N-terminus to the C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second constant domain (C2) of the TCR, where C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer of C1 and C2, and the pr first antibody has a first antigen specificity; b) allowing the host cell to express the polypeptide complex.

[017] In one aspect, the present disclosure here provides a method for producing the bispecific polypeptide complex provided herein, comprising a) introducing into a host cell a first polynucleotide encoding a first polypeptide comprising, from the N-terminal end to the C- terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the TCR, a second polynucleotide encoding a second polypeptide comprising, from the N-terminal to the C-terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, a third polynucleotide encoding a third polypeptide comprising VH of a second antibody and a fourth polynucleotide encoding a fourth polypeptide comprising VL of the second antibody, in which C1 and C2 are capable of forming a dimer comprising at least in us a non-native interlinking between C2, and the non-native interlinking is able to stabilize the dimer, and the first antibody has a first antigenic specificity and the second antibody has a second antigenic specificity; b) allowing the host cell to express the bispecific polypeptide complex.

[018] In certain embodiments, the method for producing the bispecific polypeptide complex provided herein further comprises isolating the polypeptide complex.

[019] In one aspect, the present disclosure provides a composition comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided here.

[020] In one aspect, the present disclosure here provides a pharmaceutical composition comprising the polypeptide complex provided herein, or the bispecific polypeptide complex provided here and a pharmaceutically acceptable carrier.

[021] In one aspect, the present disclosure here provides a method for treating a condition in an individual who needs it, comprising administering to the individual a therapeutically effective amount of the polypeptide complex provided herein, or the bispecific polypeptide complex provided here. In certain embodiments, the condition can be alleviated, eliminated, treated or prevented when the first antigen and the second antigen are both modulated.

[022] In certain embodiments, the non-native interchain bond is formed between a first mutated residue comprised in C1 and a second mutated residue comprised in C2. In certain embodiments, at least one of the first and second mutated residues is a cysteine residue.

[023] In certain embodiments, the non-native interchain bond is a disulfide bond.

[024] In certain embodiments, the first mutated residue is comprised within a C1 contact interface, and / or the second mutated residue is comprised within a C2 contact interface.

[025] In certain embodiments, at least one native cysteine residue is absent or present in C1 and / or C2. In certain embodiments, the native cysteine residue at position C74 of the modified CBeta is absent or present. In certain embodiments, native C74 is absent in CBeta.

[026] In certain embodiments, at least one native N-glycosylation site is absent or present in C1 and / or C2. In certain embodiments, native N-glycosylation sites are absent at C1 and / or C2.

[027] In certain embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more non-native interlinking. In certain embodiments, at least one of the non-native interchain bonds is the disulfide bond. In certain embodiments, the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more disulfide bonds.

[028] In certain embodiments, a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama.

[029] In certain embodiments, the first VH is operationally linked to C1 in a first junction domain (conjunction) and the first VL is operationally linked to C2 in a second junction domain. In certain embodiments, the first VH associates with C1 in a first junction domain via a connector, the first VL associates with C2 in a second junction domain via a connector.

[030] In certain embodiments, the first and / or the second junction domains comprise an appropriate length (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of amino acids) of the C-terminal fragment of the V / C junction of the antibody and an appropriate length (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues) of the N fragment -terminal of the V / C junction of the TCR.

[031] In certain embodiments, the modified CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of amino acid residues 9-35, 52-66, 71-86, and 122-127; and / or the modified CAlfa comprises a mutated cysteine residue within a contact interface selected from a group consisting of amino acid residues 6-29, 37- 67 and 86-95.

[032] In certain embodiments, the modified CBeta comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from: S56C, S16C, F13C, V12C, E14C, L62C, D58C, S76C and R78C and / or the modified CAlfa comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from: T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C and S62C.

[033] In certain embodiments, the modified CBeta and the modified CAlfa comprise a pair of mutated cysteine residues that replace a pair of amino acid residues selected from the group consisting of: S56C in CBeta and T49C in CAlfa, S16C in CBeta and Y11C in CAlfa, F13C in CBeta and L13C in CAlfa, S16C in CBeta and L13C in CAlfa, V12C in CBeta and S16C in CAlfa, E14C in CBeta and S16C in CAlfa, F13C in CBeta and V23C in CAlfa, L62C in CBeta and Y44C in CAlfa, D58C in CBeta and T46C in CAlfa, S76C in CBeta and T46C in CAlfa,

S56C in CBeta and L51C in CAlfa, S56C in CBeta and S62C in CAlfa, and R78C in CBeta and S62C in CAlfa, and in which the pair of cysteine residues is capable of forming a non-native disulfide bond.

[034] In certain embodiments, at least one native glycosylation site is absent or present in the modified CBeta and / or the modified CAlfa.

[035] In certain embodiments, the native glycosylation site in the modified CBeta is N69 and / or the native glycosylation site (s) in the modified CAlfa is / are selected from N34, N68 , N79 and any combination thereof.

[036] In certain embodiments, the modified CBeta does not have or maintain a FG loop that encompasses amino acid residues 101-117 of the native CBeta and / or a DE loop that encompasses amino acid residues 66-71 of the native CBeta.

[037] In certain embodiments, the modified CAlfa comprises any of SEQ ID NOs: 43-48 and / or the modified CBeta comprises any of SEQ ID NOs: 33-41 and 306.

[038] In certain embodiments, C1 comprises the modified CBeta and C2 comprises the modified CAlfa; and wherein the first junction domain comprises or is SEQ ID NO: 49 or 50, and / or the second junction domain comprises or is SEQ ID NO: 51 or 52.

[039] In certain embodiments, C1 comprises modified CAlfa and C2 comprises modified CBeta; and wherein the first junction domain comprises or is SEQ ID NO: 129 or 130, and / or the second junction domain comprises or is SEQ ID NO: 49 or 50.

[040] In certain embodiments, the modified CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35, 52-66, 71-86 and 122-127; and / or the modified CPré-Alpha comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 7-19, 26-34, 56-75 and 103-106.

[041] In certain embodiments, the modified CBeta comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from: S16C, A18C, E19C, F13C, A11C, S56C and S76C and / or CPré-Alpha The modified protein comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from S11C, A13C, I16C, S62C, T65C and Y59C.

[042] In certain embodiments, the modified CBeta and the modified CPré-Alpha comprise a pair of mutated cysteine residues that replace a pair of amino acid residues selected from the group consisting of: S16C in CBeta and S11C in CPré Alpha , A18C in CBeta and S11C in C Pre-Alpha, E19C in CBeta and S11C in C Pre-Alpha, F13C in CBeta and A13C in C Pre-Alpha, S16C in CBeta and A13C in C Pre-Alpha, A11C in CBeta and I16C in C Pre-Alpha , S56C in CBeta and S62C in C Pre-Alpha, S56C in CBeta and T65C in C Pre-Alpha, and S76C in CBeta, and Y59C in C Pre-Alpha, and in which the pair of mutated cysteine residues are capable of forming a disulfide bond non-native interchain.

[043] In certain embodiments, at least one native glycosylation site is absent in the modified CBeta and / or the modified CPré-Alpha.

[044] In certain embodiments, the glycosylation site absent or present in the modified CBeta is N69, and / or the glycosylation site absent in the modified CPré-Alpha is N50.

[045] In certain embodiments, the modified CBeta does not have or maintain a FG loop that encompasses amino acid residues 101-107 of the native CBeta and / or a DE loop in the position that encompasses amino acid residues 66-71 of the CBeta native.

[046] In certain embodiments, the modified CPré-Alpha comprises any of SEQ ID NOs: 82, 83 and 311-318; and / or the modified CBeta comprises any of SEQ ID NOs: 84, 33-41, and 319-324.

[047] In certain embodiments, C1 comprises the modified CBeta and C2 comprises the modified CPré-Alpha; and wherein the first splice domain comprises SEQ ID NO: 49 or 50 and / or the second splice domain comprises SEQ ID NO: 81 or 131.

[048] In certain embodiments, C1 comprises the modified CPré-Alpha and C2 comprises the modified CBeta; and wherein the first junction domain comprises SEQ ID NO: 132 or 133 and / or the second junction domain comprises SEQ ID NO: 49 or 50.

[049] In certain embodiments, the modified CDelta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 8-26, 43-64 and 84-88; and / or the modified gamma comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 11-35 and 55-76.

[050] In certain embodiments, the modified gamma comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from: S17C, E20C, F14C, T12C, M62C, Q57C and A19C and / or the modified CDelta comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from: F12C, M14C, N16C, D46C, V50C, F87C and E88C.

[051] In certain embodiments, the modified gamma and the modified CDelta comprise a pair of mutated cysteine residues that replace a pair of amino acid residues selected from the group consisting of: S17C in CGama and F12C in CDelta, E20C in CGama and F12C in CDelta, F14C in CGama and M14C in CDelta, T12C in CGama and N16C in CDelta, M62C in CGama and D46C in CDelta, Q57C in CGama and V50C in CDelta, A19C in CGama and F87C in CDelta, and A19C in CGama and E88C in CDelta, and in which the pair of cysteine residues introduced is capable of forming an interchain disulfide bond.

[052] In certain embodiments, at least one native glycosylation site is absent or present in the modified gamma and / or the modified CDelta.

[053] In certain embodiments, the native glycosylation site in the modified gamma is N65 and / or the native glycosylation site (s) in the modified CDelta is / are one or both of N16 and N79.

[054] In certain embodiments, the modified Gamma comprises SEQ ID NO: 113, 114, 333, 334, 335, 336, 337, 338, 339 or 340, and / or the modified CDel comprises SEQ ID NO: 115, 116, 310, 325, 326, 327, 328, 329, 330, 331 or 332.

[055] In certain embodiments, C1 comprises the modified Gamma and C2 comprises the modified CDelta; and wherein the first junction domain comprises SEQ ID NO: 117 or 118 and / or the second junction domain comprises SEQ ID NO: 119 or 120.

[056] In certain embodiments, C1 comprises the modified CDelta and C2 comprises the modified Gamma; and wherein the first junction domain comprises SEQ ID NO: 123 or 124, and / or the second junction domain comprises SEQ ID NO: 125 or 126.

[057] In certain embodiments, the first polypeptide further comprises an antibody CH2 domain and / or an antibody CH3 domain.

[058] In certain embodiments, the first antigenic specificity and the second antigenic specificity are directed to two different antigens or are directed to two different epitopes on an antigen.

[059] In certain embodiments, the first antigen-binding portion binds to CD3. In certain embodiments, the second antigen-binding portion binds to CD19. In certain embodiments, the first antigen-binding portion binds to CD19. In certain embodiments, the second antigen-binding portion binds to CD3.

[060] In certain embodiments, the first antigen-binding portion binds to CTLA-4. In certain embodiments, the second antigen-binding portion binds PD-1. In certain embodiments, the first antigen-binding portion binds to PD-1. In certain embodiments, the second antigen-binding portion binds to CTLA-4.

[061] In certain embodiments, the association is by means of a connector, a disulfide bond, a hydrogen bond, electrostatic interaction, a salt bridge or hydrophobic-hydrophilic interaction, or a combination thereof.

[062] In certain embodiments, the second antigen binding portion comprises a heavy chain variable domain and a light chain variable domain of a second antibody with the second antigen specificity.

[063] In certain embodiments, the second antigen-binding portion comprises a Fab.

[064] In certain embodiments, the first antigenic specificity and the second antigenic specificity are directed to two different antigens or are directed to two different epitopes on an antigen.

[065] In certain embodiments, one of the first and second antigenic specificities is directed at a specific T cell receptor molecule and / or a natural killer cell specific molecule (NK cell) and the other is directed at an associated antigen to tumor.

[066] In certain embodiments, one of the first and second antigenic specificities is directed at CD3 and the other is directed at a tumor-associated antigen.

[067] In certain embodiments, one of the first and second antigenic specificities is directed at CD3 and the other is directed at CD19.

[068] In certain embodiments, the first antigen binding portion further comprises a first dimerization domain and the second antigen binding portion further comprises a second dimerization domain, wherein the first and second dimerization domains are associated companies.

[069] In certain embodiments, the association is by means of a connector, a disulfide bond, a hydrogen bond, electrostatic interaction, a salt bridge or hydrophobic-hydrophilic interaction, or a combination thereof.

[070] In certain embodiments, the first and / or the second dimerization domains comprise at least a portion of an antibody hinge region, optionally derived from IgG1, IgG2 or IgG4.

[071] In certain embodiments, the first and / or the second dimerization domains further comprise a dimerization domain. In certain embodiments, the dimerization domain comprises at least a portion of an antibody hinge region, an antibody CH2 domain and / or an antibody CH3 domain.

[072] In certain embodiments, the first dimerization domain is operationally linked to the first constant region (C1) of the TCR in a third junction domain.

[073] In certain embodiments, a) C1 comprises a modified CBeta and the third junction domain is comprised in SEQ ID NO: 53 or 54; b) C1 comprises a modified CAlfa, and the third junction domain is included in SEQ ID NO: 134, 135, 140, or 141; c) C1 comprises a modified CPré-Allfa, and the third junction domain is included in SEQ ID NO: 134, 135, 140, or 141; d) C1 comprises a modified CGama and the third junction domain is included in SEQ ID NO: 121 or 122; or e) C1 comprises a

Modified CDelta and the third junction domain is included in SEQ ID NO: 127 or 128.

[074] In certain embodiments, the second dimerization domain is operationally linked to the heavy chain variable domain of the second antigen-binding portion.

[075] In certain embodiments, the first and second dimerization domains are different and are associated in a way that discourages homodimerization and / or favors heterodimerization.

[076] In certain embodiments, the first and second dimerization domains are able to associate with heterodimers through knobs-into-holes, hydrophobic interaction, electrostatic interaction, hydrophilic interaction or greater flexibility.

[077] In certain embodiments, the first antigen-binding portion comprises the first VH-containing polypeptide operably linked to a chimeric constant region, and the second polypeptide comprises VL operably linked to C2, where the chimeric constant region and C2 comprise a pair of strings selected from the group consisting of: SEQ ID NOs: 177/176, 179/178, 184/183, 185/183, 180/176, 181/178, 182/178, 184/186, 185/186 , 188/187, 196/187, 190/189, 192/191, 192/193, 195/194, 198/197, 200/199, 202/201, 203/201, 203/204, 205/204, 206 / 204, 208/207, 208/209, 211/210, 213/212, 213/151, 214/212, 214/151, 234/233, 232/231, 216/215, 218/217, 220/219 , 222/221, 224/223, 226/225, 227/223, 229/228, 229/230, 236/235 and 238/237.

[078] In certain embodiments, the first antigenicity is directed at CD3, and the first polypeptide and the second polypeptide comprise a pair of sequences selected from the group consisting of: SEQ ID NOs: 2/1, 3/4 /, 5/1, 6/3, 7/3, 9/8, 10/8, 9/11, 10/11, 13/12, 15/14, 17/16, 17/18, 20/19, 21 / 12, 65/64, 67/66, 69/68, 70/68, 70/71, 72/71, 73/71, 75/74, 75/76, 78/77, 86/85, 90/89, 91/92 /, 94/93,

96/95, 98/97, 99/95, 101/100, 101/102, 106/105, 108/107, 110/109, 112/111, 137/136, 138/136, 137/139 and 138 / 139.

[079] In certain embodiments, the first antigen-binding portion and the second antigen-binding portion comprise a combination of four sequences selected from the group consisting of: SEQ ID NOs: 12/22/24/23, 25 / 12/26/23 and 25/12/27/23, in which the first antigen-binding portion is capable of binding to CD3 and the second antigen-binding portion is capable of binding to CD19.

[080] In certain embodiments, the polypeptide complex provided here can be made into a Fab, a (Fab) 2, a diabody, a tribody, a triFabs, tandem-linked Fabs, a Fab-Fv, V-linked domains tandem, scFvs connected in tandem and among other formats.

[081] In another aspect, the present disclosure provides a kit comprising the polypeptide complex provided here for the detection, diagnosis, prognosis or treatment of a disease or condition.

[082] The foregoing features and other features and advantages of the invention will become more evident from the detailed description below of the various embodiments that proceed with reference to the attached Figures.

BRIEF DESCRIPTION OF FIGURES

[083] Figure 1 shows the chemical representations of the studied antibody formats. The T3 anti-CD3 antibody and the U4 anti-CD19 antibody were developed. The T3 constant region (CL and CH1) has been replaced by the TCR constant domains to develop a single light-heavy chain interface that is orthogonal to the regular antibody. T3 modified with TCR and native U4 together with the “knobs-into-holes” mutations in the Fc domain were used to develop the bispecific E17 and F16 antibody formats.

[084] Figures 2A-2D show overlapping representations of the antibody's Fv model and TCR structure that provide guidance for fusing the antibody's Fv and the TCR constant region. Figure 2A shows a structural Fv model of the antibody that was constructed based on the sequence of an internally developed T3 anti-CD3 antibody. Figure 2B shows the TCR structure of the PDB 4L4T. Figure 2C shows a structural Fv model of the antibody superimposed on the variable region of the TCR in different orientations. Crude chimeric proteins were created by removing the variable domain of the TCR in the overlapping representations, as shown in Figure 2D. The overlapping residues in the junction area helped to develop the junction region. The VL chain of the antibody and the alpha chain of the TCR were colored in white. The VH and beta chains were colored black.

[085] Figures 3A-3B illustrate a comparison between the TCR constant region and the antibody Fab constant region. Figure 3A illustrates a crystal structure of the TCR from PDB 4L4T. Figure 3B illustrates a structural model of the antibody Fab produced by the T3 model Fv domain and the antibody constant domain from APO 5DK3. The obvious differences in the FG and DE loops between the TCR domains and the antibody Fab domains were marked by the presentation of all side chains of the residues.

[086] Figure 4 illustrates the SDS-PAGE results of the deglycosylation mutants of chimeric antibody-TCR antibodies with the CAlfa and CBeta chains. The supernatants from all samples of the expression production in Expi293 were collected. Lanes 1, 3, 5, 7 and 9 are the non-reducing PAGEs of Design_2-QQQQ, Design_2-AAAA, Design_2-QSKE, Design_2-ASKE and Design_2-QQQQQ, respectively. Lanes 2, 4, 6, 8 and 10 are the corresponding reducing PAGEs.

[087] Figure 5 illustrates the FACS dose-dependent linkages of all binding of all deglycosylated mutants to Jurkat cells that express CD3.

Supernatants from all samples of the glycosylation mutants expressed in Expi293 were collected. The anti-wild-type CD3 antibody (T3-IgG1) was used as a positive control.

[088] Figures 6A-6B illustrate the SDS-PAGE results of the mispairing tests (incorrect pairing) of the T3 and U4 antibody chain on the IgG1 (Figure 6A) and IgG4 (Figure 6B) isotype. Lanes 1-2 are the pairs of T3_leve-U4_pesada and T3_pesada-U4_leve, respectively. Lanes 3-4 are the same pair order as lanes 1-2, but with T3 modified using the constant region of the TCR. Lanes 1-4 in both images are the unreduced samples, and lanes 5-8 are the corresponding reduced samples.

[089] Figures 7A-7B illustrate the SDS-PAGE results of the purified bispecific antibody, E17-Design_2-QQQQ in IgG1 (Figure 7A) and IgG4 (Figure 7B).

The IgG1 isotype was purified by three purification steps: protein A chromatography, ion exchange chromatography (IEC) and size exclusion chromatography (SEC).

IgG4 was obtained after two purification steps: chromatography on protein A and SEC.

Figures 7C-7D illustrate SEC-HPLC data for purified IgG1 (Figure 7C) and IgG4 (Figure 7D) samples, to determine the degree of purity of the samples.

[090] Figure 8 illustrates the SDS-PAGE results of the chimeric T3 Fab fragments with a 6xHis tag, purified by Ni SepharoseTM chromatography excel.

Lanes 1 and 3 are bands for T3-Fab-Design_2.his1, and lanes 2 and 4 are bands for T3-Fab-Design_2.his2.

[091] Figure 9 illustrates FACS dose-dependent linkages of the TCR-modified chimeric T3 Fab fragment. The monovalent form of the wild type T3 antibody (T3-Fab-IgG4) was used as a positive control. A regular human IgG4 antibody was used as a negative control.

[092] Figures 10A-10 B illustrate dose-dependent FACS linkages of the developed bispecific antibody, E17-Design_2-QQQQ, to Jurkat CD3 + cells. The wild type T3 and U4 antibodies, as well as their monovalent forms, were used as positive controls (Figure 10A) and CD19 + Ramos cells (Figure 10B).

The IgG1 and IgG4 isotypes were tested. An irrelevant human IgG1 or IgG4 antibody was used as a negative control.

[093] Figures 11A-11B illustrate the comparison of FACS bonds of two developed bispecific antibodies, E17-Design_2-QQQQ and F16-Design_2-QQQQ, to CD3 in Jurkat cells (Figure 11A) and CD19 expressed in Ramos cells (Figure 11B ). Bispecific antibodies on both IgG1 and IgG4 isotypes were tested. A regular human IgG1 or IgG4 antibody was used as a negative control.

[094] Figure 12 illustrates the cytotoxic assay for malignant B cell death directed by bispecific antibodies developed by E17-Design_2-QQQQ in both IgG1 and IgG4. Parental anti-CD3 (T3-IgG4), anti-CD19 (U4-IgG) and an irrelevant human IgG1 antibody were used as a negative control.

[095] Figure 13 compares the activity of two developed bispecific antibodies, E17-Design_2-QQQQ and F16-Design_2-QQQQ, in mediating malignant B cell death with T cell involvement. An irrelevant human IgG antibody was used as a negative control .

[096] Figures 14A-14B illustrate deconvolved mass spectra of the bispecific antibody E17-Design_2-QQQQ in non-reduced (Figure 14A) and reduced (Figure 14B) conditions. The peak at 148180.53 in Figure 14A is the correct molecular weight of the intact WuXiBody. The peak at 22877 Da indicates the light chain found in the reduced mass spectra in Figure 14B. The small peak at 149,128.45 Da in Figure 14A was deduced as the O-glycosylation (approximately (947.92 Da more) located in the light chain, as shown in Figure 14B.

[097] Figures 15A-15B illustrate the role of the interchain disulfide bond in antibody expression at the alpha / beta interface characterized by SDS-PAGE. Figure 15A illustrates the antibody containing interchain disulfide bond between CAlfa and CBeta; Figure 15B illustrates the antibody without interchain disulfide bond between CAlfa and CBeta; lanes 1 and 3 are the results of the non-reducing PAGE of Design_2-QQQQ-IgG4 with and without introduced disulfide bond, respectively. Lanes 2 and 4 are the results of Design_2-QQQQ-IgG4 reducing PAGE with and without introduced disulfide bond, respectively.

[098] Figure 16 illustrates the SDS-PAGE of the disulfide bond developed at the pre-alpha / beta interface. Lane 1 and lane 2 are “Design_5_Pre_TCR_ Conjunction’1_Cys13” and “Design_6_Pre_TCR_Conjunction’1_Cys14”, respectively, treated in non-reducing conditions. Lane 4 and lane 5 are “Design_5_Pre_TCR_Conjunction’1_Cys13” and “Design_6_Pre_TCR_Conjunction’1_Cys14", respectively, treated under reducing conditions.

[099] Figures 17A-17B illustrate SDS-PAGE of the disulfide bond developed at the delta / gamma interface. Lane 6 and lane 8 are "Design_2_Cys5_no_Glyco" and "Design_2_hypeCys2_no_Glyco", respectively. Figure 17A is the non-reducing SDS-PAGE. Figure 17B is a reducing SDS-PAGE.

[0100] Figure 18A illustrates the sequence of the native TCR alpha chain and its equivalent sequence with mutated cysteine residues. TRAC_Human is a natural sequence of the alpha chain constant region. 4L4T_Alpha_Crystal is the sequence of a crystalline structure (PDB code 4L4T) with S55C mutations that can form an interchain disulfide bond. The gray region is the constant region used as a framework for the chimeric protein in this invention.

[0101] Figure 18B illustrates the beta chain sequence of the native TCR and its equivalent sequence with mutated cysteine residues. TRBC1_Human and TRBC2_Human are natural sequences of the beta constant region.

[0102] Figure 18C illustrates the native TCR pre-alpha chain sequences.

PTCRA_Human is a natural sequence of the constant region of the pre-alpha chain (the pre-alpha chain does not only have a variable region). 3OF6_PreAlpha_Crystal is the sequence of a crystalline structure (PDB code 3OF6). The gray region is the constant region used above to define the numbering.

[0103] Figure 18D illustrates the native TCR delta chain sequences.

TRA @ _Human is the natural sequence of the delta constant region. 4LFH_Delta_Crystal is the constant region of a delta chain sequence of a crystalline structure (PDB code 4LFH). The gray region is the constant region used above to define the numbering.

[0104] Figure 18E illustrates the native TCR gamma chain sequences.

TRGC1_Human and TRGC2_Human are natural sequences of the gamma constant region.

4LFH_Gamma_Crystal is the constant region of a gamma chain sequence of a crystalline structure (PDB code 4LFH). The gray region is the constant region used above to define the numbering.

[0105] Figures 19A-19E illustrate the sequences and numbering of the TCR constant regions. Figure 19A illustrates the sequences and numbering of the TCR Alpha constant region. Figure 19B illustrates the sequences and numbering of the TCR Beta constant region. Figure 19C illustrates the sequences and numbering of the Pre-Alpha TCR constant region. Figure 19D illustrates the sequences and numbering of the TCR Delta constant region. Figure 19E illustrates the sequences and numbering of the TCR Gamma constant region.

[0106] Figures 20A-20D illustrate the sequences and numbering of the IgG1 and IgG4 knobs-into-holes. Figure 20A illustrates the sequences and numbering of IgG1 “knob” mutations. Figure 20B illustrates the sequences and numbering of IgG4 “knob” mutations. Figure 20C illustrates the sequences and numbering of IgG1 hole mutations. Figure 20D illustrates the sequences and numbering of IgG4 hole mutations.

[0107] Figures 21A-21 B illustrate the connections of E17-Design_2-QQQQ in both IgG4 (Figure 21A) and wild-type IgG1 (Figure 21B) formats to human C1Q by ELISA. A human IgG1 antibody was used as a control.

[0108] Figure 22 illustrates a schematic description of four symmetrical formats of WuXiBody, G19, G19R, G25 and G25R. For the G19 and G25 format, two chimeric Fab-type domains containing TCR were grafted to the C-terminal and N-terminal end of a normal antibody, respectively. The rectangles indicate the constant domains of the TCR and the oval indicate the variable and constant domains of an antibody. The difference between the G19 and G19R or G25 and G25R formats is the switched position of normal Fab and chimeric Fab. These formats can accommodate different variable regions of different pairs of antibodies and generally have a molecular weight around 240-250 kDa.

[0109] Figures 23A-23B illustrate the SDS-PAGE (Figure 23A) and SEC-HPLC (Figure 23B) characterizations of two purified bispecific antibodies in G19 format. The track numbers on the SDS-PAGE are consistent with the identification numbers in Figure SEC-HPLC. Lanes 1 and 2 are the antibody pair T1U6 and U6T1, respectively. In T1U6, T1 (anti-CTLA-4) was at the N-terminal end of the format, while in U6T1, U6 (anti-PD-1) was at the N-terminal end of the format. Both bispecific molecules were purified by protein A chromatography, and a purity of around 90% was obtained.

[0110] Figures 24A-24B illustrate dose-dependent FACS linkages of U6T1 and T1U6 antibodies purified in G19 format to human PD-1 cells (Figure

24A) and modified CTLA-4 (Figure 24B). An IgG4 antibody was used as a negative control.

[0111] Figures 25A-25 B illustrate the SDS-PAGE (Figure 25A) and SEC-HPLC (Figure 25B) characterizations of the bispecific antibodies purified on protein A in different symmetrical formats. Lanes 1-3 are the U6T1 antibody pair in the G19R, G25, and G25R formats, respectively. PC is a control protein known to have a molecular weight of 250 kD. All three bispecific molecules showed purity greater than 90%. The track numbers on the SDS-PAGE are consistent with the identification numbers in the SEC-HPLC Figures.

[0112] Figures 26A-26 B illustrate FACS dose-dependent binding of purified U6T1 bispecific antibodies in the G19R, G25, and G25R formats to the modified PD-1 (Figure 26A) and CTLA-4 (Figure 26B) cells. A bispecific anti-CTLA-4 x PD-1 reference antibody (BMK1.IgG1) was used as a control and an IgG4 antibody was used as a negative control.

[0113] Figures 27A-27B illustrate FACS competition assays for bispecific antibodies developed in the G19R, G25 and G25R formats to block the binding of human PD-L1 to PD-1 (Figure 27A) and binding of CD80 to CTLA-4 ( Figure 27B), respectively. A bispecific anti-CTLA-4 x PD-1 reference antibody (BMK1.IgG1) was used as a control and an IgG4 antibody was used as a negative control.

[0114] Figures 28A-28B illustrate the SDS-PAGE (Figure 28A) and SEC-HPLC (Figure 28B) characterizations of protein A purified bispecific antibodies in different symmetrical formats. Lanes 1-4 are the U6T5 antibody pair in formats G19, G19R, G25 and G25R, respectively. PC is a control protein with a molecular weight of 250 kD. All three bispecific molecules showed purity greater than 90%. The track numbers on the SDS-PAGE are consistent with the identification numbers on the SEC-HPLC Figures.

[0115] Figures 29A-29B illustrate dose-dependent FACS binding of purified bispecific antibodies in the G19, G19R, G25, and G25R formats to the modified human PD-1 (Figure 29A) and CTLA-4 (Figure 29B) cells. A bispecific anti-CTLA-4 x PD-1 reference antibody (BMK1.IgG1) was used as a control and an IgG4 antibody was used as a negative control.

[0116] Figure 30 illustrates the double bond ELISA assay of two molecules U6T5.G25 and U6T1.G25R. A bispecific anti-CTLA-4 x PD-1 reference antibody (BMK1.IgG1) was used as a control and an IgG4 antibody was used as a negative control.

[0117] Figures 31A-31B illustrate FACS competition assays for bispecific antibodies U6T5.G25 and U6T1.G25R to block the binding of human PD-L1 to PD-1 (Figure 31A) and binding of CD80 to CTLA-4 (Figure 31B), respectively. A bispecific anti-CTLA-4 x PD-1 reference antibody (BMK1.IgG1) was used as a control and an IgG4 antibody was used as a negative control.

[0118] Figure 32 illustrates the schematic description of the three symmetrical formats G26, G27 and G26R with chimeric Fab type domains with exchanged light-heavy.

[0119] Figures 33A-33B illustrate the SDS-PAGE (Figure 33A) and SEC-HPLC (Figure 33B) characterizations of protein A purified bispecific antibodies in G27 and G26R formats. Lanes 1-2 are the T4U6 antibody pair in formats G27 and G26R, respectively. Only the G26R format reached 90% purity after purification. The track numbers on the SDS-PAGE are consistent with the identification numbers in Figures SEC-HPLC.

[0120] Figures 34A-34B illustrate dose-dependent FACS linkages of the pair of bispecific T4U6 antibodies purified in the G26R format to the modified human PD-1 (Figure 34A) and CTLA-4 (Figure 34B) cells. A bispecific anti-CTLA-4 x PD-1 reference antibody (BMK1.IgG1) was used as a control and an IgG4 antibody was used as a negative control.

[0121] Figures 35A-35B illustrate the characterizations of SDS-PAGE (Figure 35A) and SEC-HPLC (Figure 35B) of the pair of bispecific U6T4 antibodies purified on protein A in the G26 format. 90% purity was obtained after purification.

[0122] Figures 36A-36D illustrate ELISA dose-dependent linkages of the G26-formatted bispecific U6T4 antibody pair to modified PD-1 (Figure 36A) and human CTLA-4 (Figure 36B) cells, as well as dose-dependent linkages by FACS of the pair of bispecific U6T4 antibodies purified in G26 format to the modified human PD-1 (Figure 36C) and CTLA-4 (Figure 36D) cells. A bispecific anti-CTLA-4 x PD-1 reference antibody (BMK1.IgG1) was used as a control and an irrelevant IgG4 antibody was used as a negative control.

[0123] Figure 37 illustrates flow cytometry histograms of the cell line transfected with cinomolg CD19 WBP701.CHO-K1.cpro1.FL.C9 and parental cell line CHO-K1.

[0124] Figure 38 illustrates the SDS-PAGE of W3438-T3U4.F16-1.uIgG4.SP. M: protein marker; Lane 1: W3438-T3U4.F16-1.uIgG4.SP, not reduced; Lane 3: W3438-T3U4.F16-1.uIgG4.SP, reduced.

[0125] Figure 39 illustrates the SEC-HPLC of W3438-T3U4.F16-1.uIgG4.

[0126] Figure 40 illustrates the SDS-PAGE of W3438-T3U4.E17-1.uIgG4.SP. M: protein marker; Lane 1: W3438-T3U4.E17-1.uIgG4.SP, not reduced; Lane 2: W3438-T3U4.E17-1.uIgG4.SP, reduced.

[0127] Figure 41 illustrates the SEC-HPLC of W3438-T3U4.E17-1.uIgG4.SP.

[0128] Figures 42A-42B illustrates the connection of W3438-T3U4.E17-1.uIgG4.SP to Ramos cells (Figure 42A) and Jurkat cells (Figure 42B) by FACS.

[0129] Figures 43A-43B illustrate the binding of W3438-T3U4.F16-1.uIgG4.SP to Ramos cells (Figure 43A) and Jurkat cells (Figure 43B) by FACS.

[0130] Figure 44 illustrates the binding of W3438-T3U4.E17-1.uIgG4.SP to a cell that expresses cynomolg CD19 by FACS.

[0131] Figure 45 illustrates the binding of W3438-T3U4.E17-1.uIgG4.SP to cinomolg CD3 by ELISA.

[0132] Figures 46A-46B illustrate the affinity of W3438-T3U4.E17-

1.uIgG4.SP to human CD19 and CD3 by measuring the binding to Ramos (Figure 46A) and Jurkat (Figure 46B) cells.

[0133] Figures 47A-47B illustrate W3438-T3U4.E17-1.uIgG4.SP binding of CD3 + cells to CD19 + cells (Figure 47A). An irrelevant IgG was used as a negative control (Figure 47B).

[0134] Figures 48A-48B illustrate the cytotoxic activity of Raji cells by T cells mediated by W3438-T3U4.E17-1.uIgG4.SP (Figure 48A) and the cytotoxic activity of Ra34 cells by T cells mediated by W3438 - T3U4.F16-1.uIgG4.SP (Figure 48B).

[0135] Figures 49A-49D illustrate the expression of CD69 and CD25 in T cells in the presence or absence of target CD19 + cells. Percentage of CD69 + expression in T cells in the subset of CD4 + T cells (Figure 49A); Percentage of CD69 + expression in T cells in the CD8 + T cell subset (Figure 49B); Percentage of CD25 + expression in T cells in the subset of CD4 + T cells (Figure 49C); Percentage of CD25 + expression in T cells in the subset of CD8 + T cells (Figure 49D).

[0136] Figures 50A-50D illustrate the release of IFN-γ and TNFα cytokines from T cells in the presence or absence of CD19 + target cells. IFNγ release in the CD4 + T cell subset (Figure 50A); TNFα release in the CD4 + T cell subset (Figure 50B); IFNγ release in the CD8 + T cell subset (Figure 50C); TNFα release in the CD8 + T cell subset (Figure 50D).

[0137] Figures 51A-51B illustrate the stability of W3438-T3U4.E17-

1.uIgG4.SP in human serum. Binding of W3438-T3U4.E17-1.uIgG4.SP samples incubated in serum to Ramos cells on the days indicated (Figure 51A); sample binding W3438-T3U4.E17-1.uIgG4.SP incubated in serum from Jurkat cells on the days indicated (Figure 51B).

[0138] Figure 52 illustrates the binding of W3438-T3U4.E17-1.uIgG4.SP to C1Q by ELISA. An IgG1 antibody was used as a control.

[0139] Figure 53 illustrates the trace of tumor volume after administration of W3438-T3U4.E17-1.uIgG4.SP at different doses in mixed humanized mice with PBMC containing Raji cell xenograft tumors. The data points represent the group mean and the error bars represent the standard error of the mean (SEM). An IgG4 antibody was used as a negative control.

[0140] Figure 54 illustrates the pharmacokinetics of W3438-T3U4.E17-1.uIgG4.SP in cinomolgo monkeys. Serum samples from two monkeys were detected by ELISA.

[0141] Figures 55A-55B illustrate the anti-drug antibody (ADA detected by ELISA) in serum samples from monkey # 1 (Figure 55A) and monkey # 2 (Figure 55B), including both pre-dose and post-dose dose of W3438-T3U4.E17-1.uIgG4.SP.

[0142] Figures 56A-56B illustrate the SDS-PAGE characterizations of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP. M: protein marker. PC: positive control of a bispecific antibody around 250 kDa (Figure 56A) and SEC-HPLC characterizations of W3248-U6T1.G25R-1.uIgG4.SP and W3248-U6T5.G25-1.uIgG4.SP (Figure 56B ).

[0143] Figure 57 illustrates the melting temperatures of W3248-U6T1.G25R-

1.uIgG4.SP, W3248-U6T5.G25-1.uIgG4.SP and a bispecific anti-CTLA-4 x PD-1 reference antibody WBP324-BMK1.uIgG1.KDL.

[0144] Figure 58 illustrates FACS connections of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to the modified human PD-1 cells. WBP324- BMK1.uIgG1.KDL, W324-BMK2.uIgG4, and W324-BMK 3.uIgG4 are different versions of the bispecific anti-CTLA-4 x PD-1 reference antibodies. WBP305-BMK1.IgG4 is an anti-PD-1 antibody. An IgG4 antibody was used as a negative control.

[0145] Figure 59 illustrates FACS connections of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to the modified cinomolgo PD-1 cells.

WBP3055_1.153.7.hAb and WBP305-BMK1.IgG4 are anti-PD-1 antibodies. An IgG4 antibody was used as a negative control.

[0146] Figure 60 illustrates FACS connections of W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to the modified human CTLA-4 cells. WBP324- BMK1.uIgG1.KDL, W324-BMK2.uIgG4, and W324-BMK 3.uIgG4 are different bispecific anti-CTLA-4 x PD-1 reference antibodies. WBP316-BMK1.IgG4 is an anti-CTLA-4-1 antibody. An IgG4 antibody was used as a negative control.

[0147] Figure 61 illustrates FACS connections from W3248-U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to modified cinomolgo CTLA-4 cells.

WBP324-BMK1.uIgG1.KDL is a bispecific anti-CTLA-4 x PD-1 reference antibody. W3162_1.154.8-z35-IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-

4. An IgG4 antibody was used as a negative control.

[0148] Figure 62 summarizes the binding affinities of W3248-U6T5.G25-

1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to CTLA-4 and PD-1, as measured by SPR. WBP316-BMK1.IgG4 is an anti-CTLA-4-1 antibody. A parental anti-PD-1 antibody was used as a control.

[0149] Figure 63 illustrates the FACS competition assays of W3248- U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to block the binding of the human PD-L1 protein to PD- cells 1 modified. WBP324-BMK1.uIgG1.KDL is a bispecific anti-CTLA-4 x PD-1 reference antibody. WBP3055_1.153.7.hAb and WBP305-BMK1.IgG4 are anti-PD-1 antibodies. An IgG4 antibody was used as a negative control.

[0150] Figure 64 illustrates the FACS competition assays of W3248- U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to block the binding of human CTLA-4 protein to modified CD80 cells . WBP324-BMK1.uIgG1.KDL is a bispecific anti-CTLA-4 x PD-1 reference antibody. W3162_1.154.8-z35- IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as a negative control.

[0151] Figure 65 illustrates FACS competition assays for W3248- U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to block the binding of cinomolgo CTLA-4 protein to modified CD80 cells . WBP324- BMK1.uIgG1.KDL is a bispecific anti-CTLA-4 x PD-1 reference antibody.

W3162_1.154.8-z35-IgG1K and WBP316-BMK1.IgG4 are anti-CTLA-4 antibodies. An IgG4 antibody was used as a negative control.

[0152] Figure 66 illustrates the double bond ELISA assay of W3248- U6T5.G25-1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP. WBP324- BMK1.uIgG1.KDL is a bispecific anti-CTLA-4 x PD-1 reference antibody. An IgG4 antibody was used as a negative control.

[0153] Figure 67 illustrates the FACS double bonding of W3248-U6T5.G25-

1.uIgG4.SP and W3248-U6T1.G25R-1.uIgG4.SP to CTLA-4 and PD-1. An IgG4 antibody was used as a negative control.

[0154] Figures 68A-68B illustrate the stability of W3248-U6T5.G25-

1.uIgG4.SP in serum for 14 days, as measured by the double binding ELISA,

CTLA-4 and PD-1 (Figure 68A) and stability of W3248-U6T1.G25R-1.uIgG4.SP in serum for 14 days, as measured by the double-binding ELISA, human CTLA-4 and PD-1 ( Figure 68B).

DETAILED DESCRIPTION OF THE INVENTION

[0155] The description of the disclosure below is merely an illustration of the various forms of carrying out the disclosure. As such, the specific changes discussed should not be construed as limiting the scope of the disclosure.

It will be apparent to a person skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments should be included in this document. All references cited here, including publications, patents and patent applications, are hereby incorporated by reference in their entirety.

[0156] Definitions

[0157] The articles “one”, “one” and “a / o (s)” are used here to refer to one or more than one (that is, at least one) grammatical object of the article. By way of example, "a polypeptide complex" means a polypeptide complex or more than one polypeptide complex.

[0158] As used in this document, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that varies up to 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% in relation to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or reference length. In certain embodiments, the terms "about" or "approximately" when preceding a numerical value indicate the value more or less a range of 15%, 10%, 5% or 1%.

[0159] Throughout this disclosure, unless the context requires otherwise, it is understood that the words "understand", "understand" and "understanding"

imply the inclusion of a declared step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consists of" is understood to include, and is limited to, anything that follows the phrase "consists of". Thus, the phrase “consists of” indicates that the elements listed are necessary or mandatory and that no other elements can be present. “Essentially consists of” the inclusion of any elements listed after the sentence and limited to other elements that do not interfere or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “essentially consists of” indicates that the elements listed are necessary or mandatory, but that other elements are optional and may or may not be present, depending on whether or not they affect the activity or action of the listed elements.

[0160] The reference throughout this disclosure to "1 (one) embodiment", "one embodiment", "a certain embodiment", "a related embodiment", "a certain embodiment" , "An additional embodiment" or "another embodiment" or combinations thereof means that a specific property, structure or feature described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the previous phrases in various places throughout this specification are not necessarily all referring to the same embodiment. In addition, the particular properties, structures or characteristics can be combined in any suitable manner in one or more embodiments.

[0161] The terms "polypeptide", "peptide" and "protein" are used interchangeably here to refer to a polymer of amino acid residues, or a set of various polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding natural amino acid, as well as to natural amino acid polymers and to a polymer of unnatural amino acids.

The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and mimetics that work in a similar way to natural amino acids.

Natural amino acids are those encoded by the genetic code, as well as those amino acids that are modified later, for example,

hydroxyproline, gamma-carboxyglutamate and O-phosphoserine.

Amino acid analogs refer to compounds that have the same basic chemical structure as a natural amino acid, that is, an alpha carbon attached to a hydrogen, a carboxyl group, an amino group and an R group, for example, homoserine, norleucine,

methionine sulfoxide, methylsulfonium methionine.

Such analogs have modified R groups (eg, norleukin) or modified peptide frameworks, but maintain the same basic chemical structure as a natural amino acid.

An alpha carbon refers to the first carbon atom that attaches to a functional group, such as a carbonyl.

A beta carbon refers to the second carbon atom attached to the alpha carbon, and the system continues to name the carbons alphabetically with Greek letters.

Amino acid mimetics refer to chemical compounds that have a different structure from the general chemical structure of an amino acid, but that work in a similar way to a natural amino acid.

The term “protein”

usually refers to large polypeptides.

The term "peptide" usually refers to short polypeptides.

Polypeptide sequences are generally described as: the left end of a polypeptide sequence is the amino terminus (N-

terminal) and the right end of a polypeptide sequence is the carboxyl terminal

(C-terminal). "Polypeptide complex", as used in this document, refers to a complex comprising one or more polypeptides that are associated to perform certain functions.

In certain embodiments, polypeptides are related to the immune system.

[0162] The term "antibody", as used in this document, encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region (“HCVR”) and a first, second and third constant region (CH1, CH2 and CH3), while each light chain consists of a variable region (“LCVR”) and a constant region (CL ).

Mammalian heavy chains are classified as α, δ, ε, γ and μ, and mammalian light chains are classified as λ or κ. The antibody has a “Y” shape, with the Y-stem consisting of the second and third constant regions of two heavy chains linked together by means of the disulfide bond. Each Y arm includes the variable region and the first constant region of a single heavy chain linked to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for binding to the antigen. The variable regions in both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs (L) include LCDR1, LCDR2 and LCDR3, heavy chain CDRs (H) include HCDR1, HCDR2, HCDR3 ). CDR limits for antibodies can be defined or identified by the Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273 (4), 927 (1997); Chothia, C. et al., J. Mol. Biol.

December 5; 186 (3): 651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196.901 (1987); Chothia, C. et al., Nature. December 21-28; 342 (6252): 877-83 (1989); Kabat E. A. et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between flanking stretches known as framework or framework regions (FRs), which are more highly conserved than the CDRs and form a structure to support the hypervariable loops. Each HCVR and LCVR comprises four FRs, and the CDRs and FRs are arranged from the amino terminal to the carboxyl terminal in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain constant regions are not involved in antigen binding, but exhibit various effector functions. Antibodies are classified into classes based on the amino acid sequence of their heavy chain constant region. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, characterized by the presence of heavy chains α, δ, ε, γ and μ, respectively.

Several of the main antibody classes are divided into subclasses such as IgG1 (heavy chain γ1), IgG2 (heavy chain γ2), IgG3 (heavy chain γ3), IgG4 (heavy chain γ4), IgA1 (heavy chain α1) or IgA2 (heavy chain α2).

[0163] The term "variable domain" in relation to an antibody, as used herein, refers to an antibody variable region or a fragment thereof comprising one or more CDRs. Although a variable domain may comprise an intact variable region (such as HCVR or LCVR), it is also possible to understand less than an intact variable region and still maintain the ability to bind to an antigen or to form an antigen binding site.

[0164] The term "antigen-binding portion", as used herein, refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs or any other antibody fragment that binds to an antigen, but does not comprise an intact native antibody structure. Examples of the antigen-binding portion include, without limitation, a variable domain, a variable region, diabody, a Fab, a Fab ', an F (ab') 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2, a bispecific dsFv (dsFv-dsFv '), disulfide-stabilized diabody (ds diabody), a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody . An antigen-binding portion is capable of binding to the same antigen to which the parental antibody binds. In certain embodiments, an antigen-binding portion may comprise one or more CDRs of a particular human antibody grafted into a structural region from one or more different human antibodies. More detailed formats of the antigen-binding portion are described in Spiess et al., 2015 (Supra) and Brinkman et al., MAbs, 9 (2), pp.182-212 (2017), which are incorporated here in its entirety.

[0165] "Fab", with respect to an antibody, refers to that portion of the antibody that consists of a single light chain (both variable and constant regions) that associate with the variable region and the first constant region of a single heavy chain by a disulfide bond. In certain embodiments, the light chain and heavy chain regions are replaced by the TCR regions.

[0166] "Fab" refers to a Fab fragment that includes a portion of the hinge region.

[0167] "F (ab ') 2" refers to a Fab' dimer.

[0168] "Diacorpo" refers to a fusion protein formed by fusing a scFv to the C-terminal end of the light chain (Fab-L-scFv) or Fd (Fab-H-scFv).

[0169] "Triacorpo" refers to a fusion protein formed by the fusion of a scFv to the light chain and the heavy chain (Fab- (scFv) 2).

[0170] A "WuXiBody" is a bispecific antibody comprising soluble chimeric protein with variable domains of an antibody and the TCR constant domains, where the subunits (such as alpha and beta domains) of the TCR constant domains are linked by modified disulfide bond .

[0171] A "difficult fragment (Fd)", relative to an antibody, refers to the amino terminal half of the heavy chain fragment that can be combined with the light chain to form the Fab.

[0172] "Fc", in relation to an antibody, refers to the portion of the antibody consisting of the second (CH2) and third (CH3) regions of a first heavy chain linked to the second and third regions of a second heavy chain via disulfide bond. The Fc portion of the antibody is responsible for several effector functions, such as ADCC and CDC, but it does not work in binding to the antigen.

[0173] "Hinge region", in terms of an antibody, includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

[0174] "CH2 domain", as used in this document, includes the portion of a heavy chain molecule that extends, for example, from amino acid 244 to amino acid 360 of an IgG antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system and amino acids 231-340, EU numbering system; see Kabat, E., et al., US Department of Health and Human Services (1983).

[0175] The "CH3 domain" extends from the CH2 domain to the C-terminal end of the IgG molecule and comprises approximately 108 amino acids. Certain classes of immunoglobulins, for example, IgM, further include a CH4 region.

[0176] "Fv", in relation to an antibody, refers to the smallest fragment of the antibody that comprises the complete antigen binding site. An Fv fragment consists of the variable domain of a single light chain linked to the variable domain of a single heavy chain. Several Fv designs have been presented, including dsFvs, in which the association between the two domains is enhanced by an introduced disulfide bond; and scFvs can be formed using a peptide linker to link the two domains together as a single polypeptide. Constructions of Fvs containing a variable domain of an immunoglobulin heavy or light chain associated with the variable and constant domain of the corresponding immunoglobulin heavy or light chain were also produced. Fvs have also been multimerized to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0177] "ScFab" refers to a fusion polypeptide with an Fd linked to a light chain by means of a polypeptide linker, resulting in the formation of a single chain Fab fragment (scFab).

[0178] "TriFabs" refers to a bispecific and trivalent fusion protein composed of three units with Fab functionality. TriFabs houses two regular Fabs fused to an asymmetric Fab-type portion.

[0179] "Fab-Fab" refers to a fusion protein formed by fusing the Fd chain of a first Fab arm to the N-terminal end of the Fd chain of a second Fab arm.

[0180] "Fab-Fv" refers to a fusion protein formed by fusing an HCVR to the C-terminal end of an Fd chain and an LCVR to the C-terminal end of a light chain. A "Fab-dsFv" molecule can be formed by introducing an interdomain disulfide bond between the HCVR domain and the LCVR domain.

[0181] "MAb-Fv" or "IgG-Fv" refers to a fusion protein formed by fusing the HCVR domain to the C-terminal end of an Fc chain and the LCVR domain, expressed separately or fused to the end C-terminal on the other, resulted in a bispecific and trivalent IgG-Fv fusion protein (mAb-Fv), with the Fv stabilized by an interdomain disulfide bond.

[0182] "ScFab-Fc-scFv2" and "ScFab-Fc-scFv" refer to a fusion protein formed by the fusion of a single chain Fab with Fc and disulfide stabilized Fv domains.

[0183] "Attached IgG" refers to a fusion protein with an Fab arm fused to an IgG to form the bispecific (Fab) 2-Fc format. It can form an "IgG-Fab" or an "Fab-IgG", with a Fab fused to the C-terminal or N-terminal end of an IgG molecule with or without a connector. In certain embodiments, the attached IgG can be further modified to an IgG-Fab4 format (see Brinkman et al., 2017, Supra).

[0184] "DVD-Ig" refers to a double variable domain antibody that is formed by the fusion of an additional HCVR domain and LCVR domain of a second specificity to an IgG heavy and light chain. “CODV-Ig” refers to a related format, in which the two HCVR domains and two LCVR domains are linked in a way that allows cross-linking of the variable domains of HCVR-LCVR, which are organized (from the N-terminal end to the C-terminal) in the order HCVRA-HCVRB and LCVRB-LCVRA, or in the order HCVRB-HCVRA and LCVRA-LCVRB.

[0185] A “CrossMab” refers to an unmodified light chain pairing technology with the corresponding unmodified heavy chain and matching the modified light chain to the corresponding modified heavy chain, thus resulting in an antibody with incorrect mismatch reduction in the light chain.

[0186] A "BiTE" is a bi-specific T cell coupling molecule, comprising a first scFv with a first antigen specificity in the LCVR-HCVR orientation linked to a second scFv with a second specificity in the HCVR-LCVR orientation.

[0187] "Percent (%) of sequence identity", relative to the amino acid sequence (or nucleic acid sequence), is defined as the percentages of amino acid residues (or nucleic acid) in a candidate sequence that are identical to of the amino acid (or nucleic acid) in a reference sequence after the alignment of the sequences and, if necessary, with the introduction of gaps to reach the maximum number of identical amino acids (or nucleic acids). Conservative substitution of amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining the percentage of amino acid sequence identity (or nucleic acid) can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available at the US National Center for Biotechnology Information (NCBI), see also, Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25: 3389-4040 (1997)) , ClustalW2 (available at the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21) : 2947-8 (2007)) and the software ALIGN or Megalign (DNASTAR). Those skilled in the art can use the standard parameters provided by the tool or can customize the parameters, as needed, for alignment, such as selecting an appropriate algorithm.

[0188] An "antigen" or "Ag", as used in this document, refers to a compound, composition, peptide, polypeptide, protein or substance that can stimulate the production of antibodies or a T cell response in cell culture or in an animal, including compositions (such as one that includes a cancer-specific protein) that are added to a cell culture (such as a hybridoma) or injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity (such as an antibody), including those induced by heterologous antigens.

[0189] An "epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent (such as an antibody) binds. Epitopes can be formed either from contiguous amino acids (also called linear or sequential epitopes) or from non-contiguous amino acids juxtaposed by the tertiary folding of a protein (also called configurational or conformational epitope). Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues in the protein, and small segments of the contiguous amino acids can be digested from an antigen binding with molecules of the main histocompatibility complex (MHC) or retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost during treatment with denaturing solvents. An epitope typically includes at least 3 and, more usually, at least 5, about 7 or about 8 to 10 amino acids in a single spatial conformation.

[0190] The term "specific binding" or "specifically binding", as used in this document, refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the polypeptide complex and the bispecific polypeptide complex provided herein specifically bind to an antigen with a binding affinity (KD) of ≤ 10-6 M (eg, ≤ 5x10-7 M, ≤ 2x10-7 M , ≤ 10-7 M, ≤ 5x10-8 M, ≤ 2x10-8 M, ≤ 10-8 M, ≤ 5x10-9 M, ≤ 2x10-9 M, ≤ 10-9 M or ≤ 10-10 M). KD, as used in this document, refers to the ratio of the dissociation rate to the association rate (koff / kon), and can be determined using surface plasmon resonance methods, for example, using an instrument like Biacore.

[0191] The term "operationally linked" or "operationally linked" refers to a juxtaposition, with or without a spacer or ligand, of two or more biological sequences of interest, so that they are in a relationship that allows them to function in the intended way. When used in relation to polypeptides, it means that the polypeptide sequences must be linked in order to allow the linked product to have the intended biological function. For example, an antibody variable region can be operationally linked to a constant region, in order to provide a stable product with antigen-binding activity. The term can also be used in relation to polynucleotides.

On the one hand, when a polynucleotide encoding a polypeptide is operationally linked to a regulatory sequence (for example, promoter, enhancer, silencing sequence, etc.) it means that the polynucleotide sequences must be linked in order to allow regulation of the polypeptide expression from the polynucleotide.

[0192] The term "fusion" or "fused", when used in relation to amino acid sequences (for example, peptide, polypeptide or protein), refers to the combination of two or more amino acid sequences, for example, by ligation chemistry or recombinant media, in a single sequence of amino acids that does not exist naturally. A fusion amino acid sequence can be produced by genetic recombination of two coding polynucleotide sequences and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.

[0193] The term "spacer", as used in this document, refers to an artificial amino acid sequence containing 1, 2, 3, 4 or 5 amino acid residues, or a length between 5 and 15, 20, 30, 50 or more amino acid residues, joined by peptide bonds and are used to link one or more polypeptides. A spacer may or may not have a secondary structure. Spacer sequences are known in the state of the art, see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al. Structure 2: 1121-1123 (1994). Any suitable spacer known in the art can be used. For example, a spacer useful in the present disclosure may be rich in glycine and proline residues. Examples include spacers with single or repeated sequences composed of threonine / serine and glycine, such as TGGGG (SEQ ID NO: 266), GGGGS (SEQ ID NO: 267) or SGGGG (SEQ ID NO: 268) or their tandem repeats ( for example, 2, 3, 4 or more repetitions). Alternatively, a spacer may be a long peptide chain containing one or more sequential or tandem repeats of the GAPGGGGGAAAAAGGGGG amino acid sequence (SEQ ID NO: 269). In a certain embodiment, the spacer comprises 1, 2, 3, 4 or more sequential or tandem repetitions of SEQ ID NO: 269.

[0194] The term "antigenic specificity" refers to a specific antigen or an epitope thereof that is selectively recognized by an antigen-binding molecule.

[0195] The term "substitution", in relation to the amino acid residue, as used in this document, refers to the natural or induced replacement of one or more amino acids by another in a peptide, polypeptide or protein. Substitution in a polypeptide can result in a decrease, increase or elimination of the polypeptide's function.

[0196] Substitution can also be "conservative substitution", in relation to the amino acid sequence, refers to the replacement of an amino acid residue with a different amino acid residue with a side chain with similar physicochemical properties or substitution of amino acids that are not critical to polypeptide activity. For example, conservative substitutions can be made between amino acid residues with non-polar side chains (e.g., Met, Ala, Val, Leu, and Ile, Pro, Phe, Trp), between residues with non-polar side chains charged (for example, Cys, Ser, Thr, Asn, Gly and Gln), between the residues with acidic side chains (for example, Asp, Glu), between the amino acids with basic side chains (for example, His, Lys and Arg), between amino acids with beta-branched side chains (eg Thr, Val and Ile), between amino acids with sulfur-containing side chains (eg Cys and Met), or between residues with aromatic side chains (eg example., Trp, Tyr,

His and Phe). In certain embodiments, substitutions, deletions or additions can also be considered as "conservative substitution". The number of amino acids that are inserted or deleted can be in the range of about 1 to 5. Conservative substitution generally does not cause significant changes in the conformational structure of the protein and, therefore, the biological activity of a protein can be maintained.

[0197] The term "mutation" or "mutated" in relation to the amino acid residue, as used in this document, refers to the replacement, insertion or addition of an amino acid residue.

[0198] As used in this document, a "homologous sequence" and "homology sequence" are used interchangeably and refer to polynucleotide sequences (or their complementary strand) or amino acid sequences that have at least 80% sequence identity (for example, at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with other strings when optionally aligned.

[0199] “T cell,” as used in this document, refers to a type of lymphocyte that plays a critical role in cell-mediated immunity, including helper or helper T cells (eg CD4 + T cells, T-type cells T helper 1, T cells type T helper 2, T cells type T helper 3, T cells type T helper 17), cytotoxic T cells (eg CD8 + T cells), memory T cells (eg cells Central memory T cells (TCM cells), effective memory T cells (TEM cells and TEMRA cells) and resident memory T cells (TRM) that are CD8 + or CD4 +), natural killer T cells (NKT) and inhibitory T cells.

[0200] A native "T cell receptor" or a native "TCR" is a heterodimeric T cell surface protein that is associated with invariant CD3 chains to form a complex capable of mediating signal transduction. The TCR belongs to the immunoglobulin superfamily and is similar to an antibody medium with a single heavy chain and a single light chain. The native TCR has an extracellular portion, a transmembrane portion and an intracellular portion. The extracellular domain of a TCR has a constant region proximal to the membrane and a variable region distal to the membrane.

[0201] The term "subject" or "individual" or "animals" or "patient", as used in this document, refers to the human or non-human animal, including a mammal or a primate, which requires diagnosis, prognosis, improvement, prevention and / or treatment of a disease or disorder. Mammals include humans, domestic animals, farm and zoo animals, sports or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears and so on .

A. Polypeptide complex

[0202] New polypeptide complexes are provided herein comprising an antibody heavy chain variable domain operably linked to a first T cell receptor constant region (TCR) and an antibody light chain variable domain operably linked to a second region TCR constant, where the first TCR constant region and the second TCR constant region are associated by at least one non-native interchain link. The polypeptide complex comprises at least two polypeptide chains, each of which comprises a variable domain derived from an antibody and a constant region derived from a TCR. The two polypeptide chains of the polypeptide complexes comprise a heavy chain variable domain pair and a light chain variable domain, which are operationally linked to a pair of TCR constant regions, respectively. Examples of TCR constant region pairs include, for example, alpha / beta, pre-alpha / beta and gamma / delta constant regions. The TCR constant regions in the polypeptide complexes provided here can be complete or in a fragment, and can be modified, as long as the TCR constant region pair is able to associate with each other to form a dimer.

[0203] It has surprisingly been found that the polypeptide complexes provided here with at least one non-native interchain bond (in particular, a non-native disulfide bond) can be expressed recombinantly and assembled in the desired conformation, which stabilizes the region dimer constant in the TCR, providing good antigen-binding activity in the variable regions of the antibody. In addition, polypeptide complexes have been found to tolerate the routine construction of antibodies well, for example, modification of glycosylation sites and removal of some natural sequences. In addition, the polypeptide complexes provided here can be incorporated in a bispecific format that can be quickly expressed and assembled with minimal or substantially nonexistent mismatch of antigen binding sequences due to the presence of the TCR constant regions in the polypeptide complexes. Additional advantages of the polypeptide complexes and constructs provided here will become clearer in the description below.

[0204] In one aspect, the present disclosure provides polypeptide complexes comprising a first polypeptide comprising, from the N-terminal to the C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first region constant (C1) of the T cell receptor (TCR), and a second polypeptide comprising, from the N-terminus to the C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, where C1 and C2 are able to form a dimer comprising at least one non-native interlink between C1 and C2, and the non-native interlink is capable of stabilizing the dimer, and the first antibody shows a first antigenic specificity.

i. TCR constant region

[0205] The polypeptide complexes provided here comprise constant regions derived from a TCR.

[0206] Native TCR consists of two polypeptide chains and generally has two types: one consists of alpha and beta chains (that is, alpha / beta TCR), and the other consists of gamma and delta chains (that is, gamma TCR / delta). These two types are structurally similar, but have different locations and functions. About 95% of human T cells have alpha / beta TCR, while the remaining 5% have gamma / delta TCR. An alpha chain precursor is also found and called a pre-alpha chain. Each of the two TCR polypeptide chains comprises an immunoglobulin domain and a region proximal to the membrane. The immunoglobulin region comprises a variable region and a constant region and is characterized by the presence of an immunoglobulin-like fold. Each TCR polypeptide chain has a cysteine residue (for example, at the C-terminal end of the constant domain or at the N-terminal end of the region proximal to the membrane) which together can form a disulfide bond that links the two TCR chains together.

[0207] Figures 18A-18E show the amino acid sequences of the native TCR constant regions of the alpha, pre-alpha, beta, gamma and delta TCR chains. For clarity and consistency, each of the amino acid residues in these sequences is numbered in Figures 19A-19E, and such numbering is used throughout this specification to refer to a particular amino acid residue from a specific TCR constant region.

[0208] The human TCR alpha chain constant region is known as TRAC, with the NCBI accession number of P01848, or an amino acid sequence of SEQ ID NO: 254.

[0209] The human TCR beta chain constant region has two different variants known as TRBC1 and TRBC2 (IMGT nomenclature), with the corresponding sequences shown in SEQ ID NO: 256 and SEQ ID NO: 257, respectively (see also Toyonaga B , et al., PNAs, Vol. 82, pp. 8624-8628, Immunology (1985)). These two beta constant domains are different in the 4th, 5th and 37th amino acid residues of exon 1. Specifically, TRBC1 has 4N, 5K and 37F in exon 1 and TRBC2 has 4K, 5N and 37Y in exon 1.

[0210] Specifically, the native TCR beta chain contains a native cysteine residue at position 74 (see Figure 19B), which is not paired and therefore does not form a disulfide bond in a native alpha / beta TCR. In certain embodiments, in the polypeptide complexes provided herein, this native cysteine residue is absent or mutated to another residue. This can be useful to avoid incorrect inter-chain or intra-chain pairing. In certain embodiments, the native cysteine residue is replaced by another residue, for example, serine or alanine. In certain embodiments, substitution in certain embodiments can improve the refolding efficiencies of the TCR in vitro.

[0211] The human TCR gamma chain regions have two variants, known as TRGC1 and TRGC2 (see Lefranc et al., Eur. J. Immunol.

19: 989-994 (1989)), with the NCBI accession number of A26659 and P03986, respectively, or the amino acid sequences of SEQ ID NO: 263 and SEQ ID NO: 265, respectively.

[0212] The human TCR delta chain constant region is known as TRDC, with the NCBI accession number of A35591, or an amino acid sequence of SEQ ID NO: 261.

[0213] The TCR constant region in the polypeptide complexes provided here can also be derived from the pre-T cell antigen receptor (pre-TCR). Pre-TCR is expressed by immature thymocytes, which play a central role in the initial development of T cells. Pre-TCR has a regular beta chain, but a special pre-alpha chain with only the constant region available, with sequence and structure distinct from those of the regular alpha chain (see Harald von Boehmer, Nat Rev Immunol, July 5; 7 (7): 571-7 (2005)). The sequence of the human pre-alpha chain constant region (PTCRA) has the NCBI accession number of AAF89556.1, or an amino acid sequence of SEQ ID NO: 259.

[0214] In the present description, the first and second TCR regions contained in the polypeptide complexes provided herein are capable of forming a dimer comprising, between the TCR constant regions, at least one non-native interchain bond which is capable of stabilizing the dimer .

[0215] The term "dimer", as used in this document, refers to an associated structure formed by two molecules, such as polypeptides or proteins, through covalent or non-covalent interactions. A homodimer or homodimerization is formed by two identical molecules and a heterodimer or heterodimerization is formed by two different molecules. The dimer formed by the first and second regions in the TCR is a heterodimer.

[0216] An interchain bond is formed between an amino acid residue in one constant region of the TCR and another amino acid residue in another constant region of the TCR. In certain embodiments, the non-native interchain bond can be any bond or interaction that is capable of associating two constant regions of the TCR in a dimer. Examples of suitable non-native interlinking include a disulfide bond, a hydrogen bond, electrostatic interaction, a salt bridge or hydrophobic-hydrophilic interaction, a knob-into-holes or a combination thereof.

[0217] A "disulfide bond" refers to a covalent bond with the R-S-S-R 'structure. The cysteine amino acid comprises a thiol group that can form a disulfide bond with a second thiol group, for example, from another cysteine residue. The disulfide bond can be formed between the thiol groups of two cysteine residues that reside respectively in the two polypeptide chains, thus forming an inter-chain bridge or an inter-chain bond.

[0218] Electrostatic interaction is a non-covalent interaction and is important in protein folding, stability, flexibility and function, including ionic interactions, hydrogen bonding and halogen bonding. Electrostatic interactions can be formed in a polypeptide, for example, between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp in the first chain and Arg, Val or Thr in the second chain.

[0219] A salt bridge are short-range electrostatic interactions that arise mainly from the anionic carboxylate of Asp or Glu and the cationic ammonium from Lys or guanidine from Arg, which are spatially proximal pairs of residues of opposite charge in native protein structures. The charged and polar residues on largely hydrophobic interfaces can act as hot spots for bonding.

Among others, residues with ionizable side chains, such as Seu, Tyr and Ser, can also participate in the formation of a salt bridge.

[0220] A hydrophobic interaction can be formed between one or more Val, Tyr and Ala in the first chain and one or more Val, Leu and Trp in the second chain, or His and Ala in the first chain and Thr and Phe in the second chain ( see Brinkmann, et al., 2017, Supra).

[0221] A hydrogen bond is formed by the electrostatic attraction between two polar groups when a hydrogen atom covalently bonds to a highly electronegative atom, such as nitrogen, oxygen or fluorine. A hydrogen bond can be formed on a polypeptide between the two-residue structural oxygen (eg, chalcogen groups) and amide hydrogen (nitrogen group), respectively, as a nitrogen group in Asn and an oxygen group in His or an oxygen group in Asn and a nitrogen group in Lys.

A hydrogen bond is stronger than a Van der Waals interaction, however, it is weaker than covalent or ionic bonds and is critical to maintaining the secondary structure and the tertiary structure. For example, an alpha helix is formed when the spacing of amino acid residues occurs regularly between the i and i + 4 positions, and a beta leaf is a stretch of the 3 to 10 amino acid peptide chain formed when two peptides join by at least two or three structural hydrogen bonds, forming a pleated and twisted sheet.

[0222] "Knobs-into-holes", as used in this document, refers to an interaction between two polypeptides, in which a polypeptide has a protuberance (that is, a "knob") due to the presence of an amino acid residue with a bulky side chain (for example, tyrosine or tryptophan) and the other polypeptide has a cavity (ie, "hole") where a small side chain amino acid residue (for example, alanine or threonine) resides, and the bulge it is positionable in the cavity to promote the interaction of the two polypeptides to form a heterodimer or complex. Methods of generating polypeptides with knobs-into-holes are known in the art, for example, as described in US Patent 5,731,168.

[0223] In certain embodiments, the dimer of the region in the TCR comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-native interchain bonds.

Optionally, at least one of the 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-native interchain bonds are disulfide bonds, hydrogen bonds, electrostatic interaction, salt bridge or hydrophobic-hydrophilic interaction, or any combination of them.

[0224] An "non-native" interlink, as used in this document, refers to an inter-link that is not found in a native association of the regions in the TCR of the native equivalent. For example, a non-native interchain bond can be formed between a mutated amino acid residue and a native amino acid residue, each residing in a respective region on the TCR; or, alternatively, between two mutated amino acid residues that reside in the TCR regions respectively. In certain embodiments, at least one non-native interchain bond is formed between a first mutated residue comprised in the first constant region of the TCR and a second mutated residue comprised in the second constant region of the TCR of the polypeptide complex.

[0225] An "mutated" amino acid residue refers to one that is replaced, inserted or added and is different from its native equivalent residue in a corresponding constant region of the native TCR. For example, if an amino acid residue at a certain position in the TCR constant region of the wild type is referred to as the “native”, then its equivalent mutant is any residue that is different from the native residue, but that resides in the same position in the TCR region.

A mutated waste can be a different waste that replaces the native waste in the same position or that is inserted before the native waste and therefore assumes its original position.

[0226] In certain embodiments, the mutated residue may be a natural amino acid residue. In certain embodiments, at least one of the first and second non-native amino acid residues is a mutated cysteine residue. In certain embodiments, one or more of the non-native interchain bonds is a disulfide bond. In certain embodiments, the non-native disulfide bond can be formed between two mutated cysteine residues comprised in the first and second regions of the TCR, respectively.

[0227] In certain embodiments, at least one of the first and second mutated residues is an unnaturally occurring amino acid residue.

An unnaturally occurring amino acid residue refers to an amino acid residue that is not found naturally in human proteins, but can be expressed by means of a nucleic acid codon that can be incorporated into the coding polynucleotide. For example, unnatural amino acids, such as L-3,4-dihydroxyphenylalanine (L-DOPA) can react and cross-link with natural amino acids, such as cysteine, histidine and lysine, through periodate-induced oxidation. It has been shown that by incorporating L-DOPA into an antibody, the unnaturally occurring amino acid was able to cross-link effectively with residues in the antigen, resulting in a covalently linked antigen-antibody complex (Xu, J. et al., 2014 , Structure-based non-canonical amino acid Design to covalently crosslink an antibody-antigen complex.Journal of Structural Biology, 185 (2), pp.215-222). It is considered in this document that the mutated amino acid residue in the first and / or second regions contained in the TCR may comprise an unnaturally occurring amino acid residue, such as L-DOPA, which can cross-link with a natural amino acid residue ( or, alternatively, an unnaturally occurring amino acid residue) to form a non-native inter-chain covalent bond.

[0228] In certain embodiments, at least one non-native disulfide bond is formed between a mutated cysteine residue and a native cysteine residue. In certain embodiments, non-native disulfide bonds are formed between two mutated cysteine residues. In certain embodiments, at least one of the cysteine residues that form the non-native disulfide bond is a mutated cysteine residue. In certain embodiments, both cysteine residues that form the non-native disulfide bond are mutated cysteine residues in the first and second regions of the TCR, respectively.

[0229] In certain embodiments, the first and / or second regions contained in the TCR can be modified to comprise one or more mutated amino acid residues that are responsible for forming the non-native interchain bond. To introduce such a mutated residue into the TCR constant region, a coding sequence for a TCR region can be modified to, for example,

replace a codon that encodes a native residue with the codon that encodes the mutated residue, or to insert a codon that encodes the mutated residue before the codon of the native residue. One or more desired mutated amino acid residues can be introduced into the TCR constant region, for example, one or more amino acid residues (for example, cysteine residue) capable of forming a disulfide bond, which can lead to electrostatic interactions between the two TCR constant regions, which can increase the flexibility of the TCR constant regions, which position at least one of the covalent bond-forming amino acids away from the TCR constant domain, such as a hydrogen bond, which can contribute to the formation of a salt; hydrophobic amino acid residues capable of leading to hydrophobic interactions; and hydrophilic amino acid residues capable of leading to hydrophilic interactions, and so on.

[0230] In certain embodiments, the first and / or second regions contained in the TCR can be modified to comprise one or more mutated cysteine residues. For example, a non-cysteine residue can be replaced by a cysteine residue or a cysteine residue can be inserted between two native adjoining native cysteine residues. The substitution positions can be determined in such a way that, after the replacement of the cysteine residues, a non-native disulfide interchain bond could be formed between the two regions contained in the TCR. For this purpose, several factors can be considered, including, for example, the cysteine residues that form the disulfide bond may be close enough, may have adequate alpha-beta binding orientation, the thiol groups of the cysteine residues may be targeted in order to face each other, the residue to be replaced may have a side chain with chemical property relatively similar to that of cysteine and / or the substitution would not substantially disturb the tertiary structure of the TCR constant region or the polypeptide complex itself.

[0231] A person skilled in the art can determine the distance and angle between two amino acid residues to be replaced using suitable methods known in the art, for example, without limitation, distance maps by photodetection, computational modeling, spectroscopy by NMR or X-ray crystallography. In an illustrative example, for a polypeptide of interest (such as a TCR constant region), its crystalline protein structure can be obtained from public databases, such as a PDB database, or alternatively be elucidated using methods such as crystallography X-ray. Suitable software can be used to determine the distances and angles between the amino acid residues based on the crystalline protein structure data. In certain embodiments, in the polypeptide complex provided here, a disulfide bond can be formed between mutated cysteine residues with the respective beta carbons close enough, for example, at a distance of less than 8 angstroms, 7 angstroms, 6 angstroms, 5 angstroms , 4 angstroms, 3 angstroms, 2 angstroms, 1 angstrom or less when the complex is folded correctly.

[0232] Additional positions suitable for the development of the first and / or second regions in the TCR can be obtained from published crystal structure data on the complex between alpha and beta TCR (Boulter, JM et al., Protein engineering, 16 (9), pp.707-711 (2003)), or gamma and delta (Allison, TJ et al., Nature, 411 (6839), pp.820-824. (2001); Uldrich, AP et al. , Nature Immunology, 14 (11), pp. 137-1145 (2013)). Once the residue to be replaced is determined, a technician on the subject can easily identify the codon of interest to be mutated (for example, by aligning sequences using existing software, such as ClustalW (electronic address of the European Bioinformatics Institute (www .ebi.ac.uk / index.html)), and then mutate it into the cysteine codon by methods known in the art, such as PCR mutagenesis.

[0233] The formation of the interchain disulfide bond can be determined by suitable methods known in the art. For example, the expressed protein product can be subjected to reducing and non-reducing SDS-PAGE, respectively, followed by comparison of the resulting bands to identify the potential difference that indicates the presence of interchain disulfide bond.

[0234] Non-native interlinking is able to stabilize the polypeptide complex. Such effects on stabilization can be achieved in several ways. For example, the presence of the mutated amino acid residue or non-native interchain linkage can allow the polypeptide complex to be expressed in a stable manner and / or to be expressed at a high level and / or to associate in a stable complex with the desired biological activity (for example, antigen binding activity) and / or express and organize at a high level of desired stable complex with the desired biological activity. The ability of the interchain bond to stabilize the first and second regions contained in the TCR can be assessed using appropriate methods known in the art, such as the molecular weight presented in SDS-PAGE, or thermostability measured by differential scanning calorimetry (DSC ) or differential scanning fluorimetry (DSF). In an illustrative example, the formation of a stable polypeptide complex provided here can be confirmed by SDS-PAGE, if a product illustrates a molecular weight comparable to the combined molecular weight of the first and the second polypeptides. In certain embodiments, the polypeptide complex provided here is stable, as its thermal stability is not less than 50%, 60%, 70%, 80% or 90% of that of a natural Fab. In certain embodiments, the polypeptide complex provided here is stable, as its thermal stability is comparable to that of a natural Fab.

[0235] Without being bound by any theory, it is believed that the non-native interchain bond (such as a disulfide bond) formed between the first and second TCR constant regions in the polypeptide complexes is capable of stabilizing the heterodimer of the TCR constant regions thus increasing the correct folding level, structural stability and / or the expression level of the heterodimer and the polypeptide complexes. In contrast to the native TCR anchored in the T cell surface membrane, it was found that the heterodimers of native extracellular TCR domains are much less stable, despite their similarity to the antibody Fab in terms of the three-dimensional structure. In fact, the instability of the native TCR in soluble condition used to be a significant obstacle that prevented the elucidation of its crystalline structure (see Wang, Protein Cell, 5 (9), pp.649-652 (2014)). By introducing a pair of cysteine mutations (Cys) in the regions contained in the TCR and, thereby allowing the formation of non-native interchain disulfide bond, polypeptide complexes can be expressed in a stable manner, while at the same time, the capacity binding to the antigen of the antibody variable region is maintained.

[0236] The TCR constant region that comprises a mutated residue is also referred to here as a "modified" TCR constant region. In certain embodiments, the first TCR constant region (C1) of the polypeptide complex comprises a modified TCR alpha chain (CAlfa) and the second TCR constant region (C2) of the TCR comprises a modified TCR beta chain (CBeta). In certain embodiments, C1 comprises a modified CBeta and C2 comprises a modified CAlfa. In certain embodiments, C1 comprises a modified TCR Pre-alpha chain (C Pre-Alpha), and C2 comprises a modified CBeta. In certain embodiments, C1 comprises a modified CBeta, and C2 comprises a modified CPre-Alpha. In certain embodiments, C1 comprises a modified TCR gamma chain (Gamma) and C2 comprises a modified TCR delta chain (CDelta). In certain embodiments, C1 comprises a modified CDelta and C2 comprises a modified Gamma.

[0237] In certain embodiments, the modified TCR constant region comprises one or more mutated cysteine residues. In certain embodiments, the one or more mutated residues are comprised within a contact interface of the first and / or second modified TCR constant regions.

[0238] The term “contact interface”, as used in this document, refers to the specific region (s) in the polypeptides where the polypeptides interact / associate. A contact interface comprises one or more amino acid residues that are capable of interacting with the corresponding amino acid residue (s) that come into contact or in association when the interaction occurs.

The amino acid residues in a contact interface may or may not be in a consecutive sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface can be separated at different positions in the linear sequence.

[0239] In certain embodiments, the modified CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35, 52-66, 71-86, and 122-127 . In certain embodiments, the modified CAlfa comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 6-29, 37-67 and 86-95. Unless specified, the numbering donates amino acid residues in the TCR constant region in the present disclosure is as defined below in Figures 19A-19E.

[0240] In certain embodiments, one or more disulfide bonds can be formed between the modified CAlfa and the modified CBeta. The CBeta-mutated cysteine residue can be a selected substitution group consisting of: S56C, S16C, F13C, V12C, E14C, F13C, L62C, D58C, S76C, and R78C, and / or the CAlfa mutated cysteine residues can be a selected substitution from the group consisting of: T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C and S62C.

In certain embodiments, the pair of mutated cysteine residues may be a pair of substitutions selected from the group consisting of: S16C in CBeta and Y11C in CAlfa, F13C in CBeta and L13C in CAlfa, S16C in CBeta and L13C in CAlfa, V12C in CBeta and S16C in CAlfa, E14C in CBeta and S16C in CAlfa, F13C in CBeta and V23C in CAlfa, L62C in CBeta and Y44C in CAlfa, D58C in CBeta and T46C in CAlfa, S76C in CBeta and T46C in CAlfa, S56C in CAlfa, S56C in CBeta and T49C in CAlfa, S56C in CBeta and L51C in CAlfa, S56C in CBeta and S62C in CAlfa, and R78C in CBeta and S62C in CAlfa and in which the pair of cysteine residues is capable of forming a non-native disulfide bond.

[0241] As used throughout the order, “XnY” with respect to a TCR constant region means that the amino acid residue number X in the TCR constant region (based on the numbering in Figures 19A-19E, as shown here ) is replaced by the amino acid residue Y, where X and Y are the abbreviation for a letter of a specific amino acid residue respectively.

Note that the number n is based solely on the numbering provided in Figures 19A-19E, and may look different from its actual position. To illustrate, the CBeta (S56C) (N69Q) sequence shown in SEQ ID NO: 34 is used as an example. While the substitution from S to C occurs at the 48th residue in SEQ ID NO: 34, the same residue is designated as the 56th residue based on the numbering system shown in Figures 19A-19E, and therefore, this substitution of S to C is designated as S56C, but not S48C. Likewise, the substitution from N to Q is also designated as N69Q based on the numbering system shown in Figures 19A-19E. This amino acid residue substitution designation rule applies to the entire region contained in the TCR in this disclosure, unless otherwise specified. Likewise, “XnY”, when used with respect to an Fc region, means that the n ° amino acid residue X in the constant region of Fc (based on the numbering in Figures 20A-20D, as shown here) is replaced by the residue of amino acid Y.

[0242] In certain embodiments, the modified CBeta comprises or is any of SEQ ID NOs: 33-41, and the modified CAlfa comprises or is any of SEQ ID NOs: 43-48.

[0243] In certain embodiments, one or more non-native disulfide bonds can be formed within the contact interfaces between CPré-Alpha and CBeta.

In certain embodiments, the CPré-Alpha contact interface is selected from substitutions in the position of amino acid residues 7-19, 26-34, 56-75 and 103-

106. In certain embodiments, the CBeta contact interface is selected from substitutions in the amino acid residue position 9-35, 52-66, 71-86 and 122-

127.

[0244] In certain embodiments, one or more disulfide bonds can be formed between the modified TCR Pre-Alpha constant region (C Pre-Alpha) and the beta chain constant region (CBeta). CBeta-mutated cysteine residues may be a selected substitution from the group consisting of: S16C, A18C, E19C, F13C, A11C, S56C and S76C and / or CPré-Alpha mutated cysteine residues may be a selected substitution from the group consisting of: S11C, A13C, I16C, S62C, T65C and Y59. In certain embodiments, the pair of mutated cysteine residues can be a pair of substitutions selected from the group consisting of: S16C in CBeta and S11C in C Pre-Alpha, A18C in CBeta and S11C in C Pre-Alpha, E19C in CBeta and S11C in C Pre-Alpha, F13C in CBeta and A13C in C Pre-Alpha, S16C in CBeta and A13C in C Pre-Alpha, A11C in CBeta and I16C in C Pre-Alpha, S56C in CBeta and S62C in C Pre-Alpha, S56C in CBeta and T65C in CPré-Alpha, and S76C in CBeta, and Y59C in CPré-Alpha, and where the pair of mutated cysteine residues are capable of forming a non-native disulfide bond.

[0245] In certain embodiments, the modified CBeta comprises or is any of SEQ ID NOs: 33-41, and the modified CPré-Alpha comprises or is any of SEQ ID NOs: 82 and 83.

[0246] In certain embodiments, one or more non-native disulfide bonds can be formed within the contact interfaces between CGama and CDelta.

In certain embodiments, the Gamma contact interface is selected from substitutions in the position of amino acid residues 11-35 and 55-76. In certain embodiments, the CDelta contact interface is selected from substitutions in the position of amino acid residues 8-26, 43-64 and 84-88.

[0247] In certain embodiments, one or more disulfide bonds can be formed between the gamma and the modified CDelta. The gamma mutated cysteine residue can be a selected substitution from the group consisting of: S17C, E20C, F14C, T12C, M62C, Q57C, and A19C, and / or the CDelta mutated cysteine residues can be a selected substitution from the group consisting of: F12C, M14C, N16C, D46C, V50C, F87C and E88C. In certain embodiments, the pair of mutated cysteine residues may be a pair of substitutions selected from the group consisting of: S17C in CGama and F12C in CDelta, E20C in CGama and F12C in CDelta, F14C in CGama and M14C in CDelta, T12C in CGama and N16C in CDelta, M62C in CGama and D46C in CDelta, Q57C in CGama and V50C in CDelta, A19C in CGama and F87C in CDelta, and A19C in CGama and E88C in CDelta, and where the pair of cysteine residues introduced is capable of forming an interchain disulfide bond.

[0248] In certain embodiments, the modified Gamma comprises or is any of SEQ ID NOs: 113 and 114, and the modified CDelta comprises or is any of SEQ ID NOs: 115 and 116.

[0249] In addition to the non-native amino acid residue, the modified TCR constant region, in certain embodiments, may further comprise a further modification of one or more native residues following the wild-type TCR constant region. Examples of such additional modifications include, such as modifying a native cysteine residue, modifying a native glycosylation site and / or modifying a native loop.

[0250] Certain regions of native TCR (such as CBeta) comprise a native cysteine residue that, in some embodiments of the present disclosure, can be modified (for example, removed) or, alternatively, can be maintained in some others embodiments. In certain embodiments, a native disulfide bond in the alpha / beta heterodimeric TCR between the TRAC and TRBC1 or TRBC2 constant domain, that is, between TRAC exon 2 Cys4 and TRBC1 or TRBC2 exon 2 Cys2, according to IMGT nomenclature of TCR, may be present or absent.

[0251] In certain embodiments, at least one native cysteine residue is absent or present in the modified CBeta. For example, the native cysteine residue at position C74 of CBeta may be present or absent in the modified CBeta. In certain embodiments, the modified CBeta, in which the native C74 cysteine residue is absent, comprises or is any of SEQ ID NOs: 32-41.

[0252] Without being bound by any theory, it is believed that the polypeptide complex provided here is advantageous, as it tolerates the presence and absence of the native cysteine residue in CBeta. Although it has been suggested (see, for example, US Patent No. 7,666,604) that the presence of native cysteine residues in soluble TCR heterodimers is detrimental to the TCR's ability to bind to the ligand, the polypeptide complex provided here may tolerate the presence of this native cysteine residue without negatively affecting its antigen-binding activity. In addition, the polypeptide complex provided here, in the absence of the native cysteine residue, expressed at high levels, despite the contrary teachings of Wu et al. mAb, 7 (2), pp.364-376 (2005) that the native disulfide bond in the TCR heterodimer is good for stabilizing the TCR heterodimer.

[0253] In certain embodiments, one or more native glycosylation sites present in the regions contained in the native TCR can be modified (e.g., removed) or maintained in the polypeptide complex shown in the present disclosure. The term "glycosylation site", as used in this document, with respect to a polypeptide sequence, refers to an amino acid residue with a side chain to which a carbohydrate moiety (for example, an oligosaccharide structure) can be attached . Glycosylation of polypeptides as antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence , such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the binding of one of the N-aceylgalactosamine, galactose or xylose sugars to a hydroxy-amino acid, most commonly serine or threonine. Removal of native glycosylation sites can conveniently be accomplished by changing the amino acid sequence so that one or more of the tripeptide sequences described above (for the N-linked glycosylation sites) or one or more serine or threonine residues ( for O-linked glycosylation sites) are replaced.

[0254] In certain embodiments, in the polypeptide complex provided here, at least one native glycosylation site is absent or present in the modified regions of the TCR, for example, in the first and / or second regions of the TCR. Without being linked to any theory, it is believed that the polypeptide complex provided here can tolerate the removal of all or part of the glycosylation sites without affecting the expression and stability of the protein, contrary to the existing teachings that show that the presence of sites N-linked glycosylation in the TCR constant region, such as CAlfa (eg N34, N68 and N79) and CBeta (eg N69), is required for protein expression and stability (see Wu et al., Mabs, 7: 2, 364-376, 2015).

[0255] In certain embodiments, in the polypeptide complex provided here, at least one of the N-glycosylation sites on the modified CAlfa, for example, N34, N68, N79 and N61, are absent or present. In certain embodiments, the modified CAlfa sequences absent from a glycosylation site comprise or are any of SEQ ID NOs: 44-48. In certain embodiments, at least one of the N-glycosylation sites on the modified CBeta, for example, N69, is absent or present. The modified CBeta sequences (TRBC1) absent from the glycosylation site comprise or are any of SEQ ID NOs: 34-36. Modified CBeta sequences (TRBC2) absent from a glycosylation site comprise or are any of SEQ ID NOs: 38-

40.

[0256] In certain embodiments, in the polypeptide complex provided here, at least one of the N-glycosylation sites on the modified CPré-Alpha, for example, N50, is absent or present. The modified CPré-Alpha sequence absent from a glycosylation site comprises or is SEQ ID NO: 83.

[0257] In certain embodiments, in the polypeptide complex provided here, at least one of the N-glycosylation sites in the modified gamma, for example, N65, is absent or present. In certain embodiments, the modified gamma sequence absent from a glycosylation site comprises or is SEQ ID NO: 114. In certain embodiments, at least one of the N-glycosylation sites on the modified CDelta, for example, N16 and N79, is absent or present. The modified CDelta sequence absent from the glycosylation site comprises or is SEQ ID NO: 116.

[0258] In certain embodiments, one or more native secondary structures present in the regions contained in the native TCR can be modified (for example, removed) or maintained in the polypeptide complex shown in the present disclosure. In certain embodiments, a native loop (such as the FG loop and / or the DE loop of the native CBeta) is modified (e.g., removed) or maintained in the polypeptide complex provided here. The term “FG loop” and “DE loop” are structures found mainly in the constant domain of the beta chain of the TCR.

The FG loop encompasses amino acid residues 101-117 of native CBeta and is a structural element exposed to an unusually elongated solvent that forms a component of an alpha / beta TCR cavity against CD3. The DE loop covers amino acid residues 66-71 of the native CBeta. An alignment of the sequence of the constant region of the beta chain of the TCR with the constant region of the immunoglobulin CH1 revealed that the FG loop of the constant region of the beta chain of the TCR is significantly longer. Figure 3 illustrates the differences in the constant regions between the T cell beta chain and the antibody heavy chain. In certain embodiments, the sequence in the FG loop (YGLSENDEWTQDRAKPVT, SEQ ID NO: 79) is missing and / or is replaced by YPSN (SEQ ID NO: 80). In certain embodiments, the sequence in the native DE loop (QPALNDSR, SEQ ID NO: 88) is missing and / or is replaced by QSGR (SEQ ID NO: 87). In certain embodiments, the CBeta sequences absent from the native FG loop comprise or are any of SEQ ID NOs: 37-40. In certain embodiments, the CBeta sequence missing from the native FG loop and native DE loop comprises or is SEQ ID NO: 41.

[0259] In the polypeptide complex provided here, constant regions derived from a TCR are operably linked to variable regions derived from an antibody. The variable region of the heavy chain or the light chain of an antibody can be operationally linked to a constant region of the TCR, with or without a spacer.

[0260] In certain embodiments, the first antibody variable domain (VH) is fused to the first constant region (C1) of the TCR in a first junction domain, the first antibody variable domain (VL) is fused to the second constant region (C2) of the TCR in a second junction domain.

[0261] "Junction domain", as used in this document, refers to a boundary or boundary region in which two amino acid sequences are fused or combined. In certain embodiments, the junction domain comprises at least a portion of the C-terminal fragment of a first fusion partner fused to at least a portion of the N-terminal fragment of a second fusion partner, with or without a spacer between they. In such embodiments, the junction domain comprises fragments from both melting partners and the melting point resides at the point where the two fragments connect to each other, for example, directly or through a spacer. In certain other embodiments, the join domain consists of a fragment from a fusion partner.

In such embodiments, the melting point can be at either end of the junction domain.

[0262] In certain embodiments, the first junction domain comprises at least a portion of the C-terminal fragment of a V / C junction of the antibody, and at least a portion of the N-terminal fragment of a V / C junction of the TCR.

[0263] The term "antibody V / C junction", as used in this document, refers to the boundary of the variable domain and the constant domain of the antibody, for example, the boundary between the variable domain of the heavy chain and the CH1 domain , or between the variable domain of the light chain and the constant domain of the light chain.

Likewise, the term “TCR V / C junction” refers to the limit of the variable domain and the constant domain of the TCR, for example, the limit between the variable domain and the constant domain of the TCR Alpha, or between the domain variable and the constant domain of the TCR Beta.

[0264] If the Fv region of an immunoglobulin is aligned with an immunoglobulin-like TCR domain, the antibody V / C junction and the TCR V / C junction are also aligned. An example is shown in Table 1 below, where the V / C junction of the antibody heavy chain (SEQ ID NO: 270) is aligned with the V / C junction of TCR Beta (SEQ ID NO: 271) and the V / C junction of the antibody light chain (SEQ ID NO: 272) is aligned with the V / C junction of the TCR Beta (SEQ ID NO: 273).

[0265] The first and / or second junction domains of the polypeptide complex, as provided herein, can be selected in such a way that it comprises an appropriate length (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues) of the C-terminal fragment of the V / C junction of the antibody and an appropriate length (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues) of the N-terminal fragment of the V / C junction of the TCR. For example, as shown in Table 1, the junction domain can be selected so as to have the entire sequence of the TCR V / C junction (see, for example, SEQ ID NO: 145) or most of the sequence (see , for example, SEQ ID NO: 147) or some sequence (see, for example, SEQ ID NO: 146) of the V / C junction of the TCR. Still using Table 1 as an example, the junction domain can comprise more residues from the V / C junction of the TCR than from the V / C junction of the antibody (see, for example, SEQ ID NO: 147) or vice versa (see , for example, SEQ ID NO: 146)

[0266] In certain embodiments, the first and / or the second junction domains of the polypeptide complex, as provided herein, have a total length comparable to that of the antibody V / C junction or the TCR V / C junction.

[0267] An appropriate join domain can be determined on a structural basis. For example, the three-dimensional structures of the antibody and TCR can be superimposed, and overlaps of the antibody V / C junction and the TCR V / C junction on the overlapped structure can be determined and considered when determining the length or proportion of sequences from the V / C junction of the antibody or TCR.

[0268] In certain embodiments, the first and / or the second junction domains comprise a spacer between the V / C junction fragments of the antibody and V / C of the TCR. Any suitable sequences or length of spacer sequences can be used, as long as it does not negatively affect the binding to the antigen or the stability of the polypeptide complex.

[0269] Examples of variable domain limit / antibody constant limit and variable domain limit / TCR constant sequence and antibody variable region limit / TCR constant are provided in Tables 1-6 below.

[0270] In certain embodiments, C1 comprises a modified CBeta and C2 comprises a modified CAlfa. To illustrate, Table 1 illustrates examples of designs for the junction domains useful for the antibody VH fused to TCR CBeta, or for the antibody VL fused to TCR CAlfa. The limit of the antibody constant / VH domain is aligned with the limit of CBeta / TCR variable, and the limit of constant domain / VL of antibody is aligned with the limit of CAlfa / TCR variable. Examples of drawings of the junction domains are also presented in the form of alignment (see, for example, SEQ ID NO: 144, 145, 146, or 147), where the first or the second junction domain is represented in underlined form. In such embodiments, the first junction domain comprises or is SEQ ID NO: 49 or 50. In such embodiments, the second junction domain comprises or is SEQ ID NO: 51 or 52.

TABLE 1. Drawings of the first and second junction domains for VH-CBeta / VL-CAlfa Junction Junction (heavy chain) (light chain) Constant Variable Constant Variable Antibody_VH …… LVTVSS AS - TKGPS …… Antibody_VL …… KVEIK RTVAAPSVF …… SEQ ID NO: 270 SEQ ID NO: 272

TCR_beta …… RLTVLE DLKNVFPPE …… TCR_alpha …… KLIIK PDIQNPDPA …… SEQ ID NO: 271 SEQ ID NO: 273 H_Conjunction_1 …… LVTVSS ASKNVFPPE …… L_Conjunction_1 …… KVEIK RTVAAPDPA …… SEQ IDunction …… … LVTVLE DLKNVFPPE …… L_Conjunction_2 …… KVEIK PDIQNPDPA …… SEQ ID NO: 145 SEQ ID NO: 147

[0271] In certain embodiments, C1 comprises a modified CAlfa and C2 comprises a modified CBeta. Table 2 illustrates the examples of designs for the junction domains useful for the TCR CAlfa fused antibody VH, or for the TCR CBeta fused antibody VL. The limit of the antibody constant / VH domain is aligned with the limit of CAlfa / TCR variable and the limit of constant domain / VL of antibody is aligned with the limit of CBeta / TCR variable.

Examples of drawings of the junction domains are also presented in the form of alignment (see, for example, SEQ ID NO: 148, 149 or 150), where the first or the second junction domain is represented in underlined form. In such embodiments, the first junction domain comprises or is SEQ ID NO: 129 or 130. In such embodiments, the second junction domain comprises or is SEQ ID NO: 49 or 50.

TABLE 2. Drawings of the first and second junction domains for VH-CAlfa / VL-CBeta Junction Junction (heavy chain) (light chain) Constant Variable Constant Variable Antibody_H …… LVTVSS --ASTKGPS …… Antibody_L …… KVEIK-- RT- --VAAPSVF …… SEQ ID NO: 274 SEQ ID NO: 276 TCR_alpha …… KLIIK-- PDIQNPDPA …… TCR_beta …… RLTVLE -DLKNVFPPE …… SEQ ID NO: 275 SEQ ID NO: 277 H_Conjunction_3 L_Conjunction_3… LTV ASIQNPDPA …… -DLKNVFPPE …… SEQ ID NO: 148 SEQ ID NO: 150 H_Conjunction_4 L_Conjunction_4 …… KVEIKLE …… LVTVSS PDIQNPDPA …… -DLKNVFPPE …… SEQ ID NO: 149 SEQ ID NO: 150

[0272] In certain embodiments, C1 comprises a modified CBeta and C2 comprises a modified CPré-Alpha. Table 3 illustrates the examples of designs for the junction domains useful for the VH of antibody fused to CBeta of TCR, or for the VL of antibody fused to CPré-Alpha of TCR. The limit of the antibody constant / VH domain is aligned to the limit of CBeta / TCR variable, and the limit of the antibody constant / VL domain is aligned to the limit of

C Pre-Alpha / TCR variable. Examples of drawings of the junction domains are also presented in the form of alignment (see, for example, SEQ ID NO: 170, 171, 169 or 156), where the first or the second junction domain is represented in underlined form. In such embodiments, the first junction domain comprises or is SEQ ID NO: 49 or 50. In such embodiments, the second junction domain comprises or is SEQ ID NO: 81 or 131.

TABLE 3. Drawings of the first and second junction domains for VH-CBeta / VL-C Pre-Alpha Junction Junction (heavy chain) (light chain) Constant Variable Constant Variable Antibody_H …… LVTVSS AS - TKGPS …… Antibody_L …… KVEIK RTVAAPSVF …… SEQ ID NO: 283 SEQ ID NO: 285 TCR_beta …… RLTVLE DLKNVFPPE …… TCR_alfa …… KLIIK PDIQNPDPA …… SEQ ID NO: 284 SEQ ID NO: 286 Pre-TCR_alfa …… GCPAL PTGVGGTPF …… SEQ ID NO: 287 H_Conjunction_A …… LVTVSS ASKNVFPPE …… L_Conjunction_A …… KVEIK RTVAAGTPF …… SEQ ID NO: 170 SEQ ID NO: 169 H_Conjunction_B …… LVTVLE DLKNVFPPE …… L_Conjunction_B …… KVEIK PTG: NO ID: 156

[0273] In certain embodiments, C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta. Table 4 illustrates the examples of designs for the junction domains useful for the VCR of antibody fused to CPré Alpha of TCR, or for the VL of antibody fused to CBeta of TCR. The boundary of the antibody constant / VH domain is aligned with the boundary of CPré Alpha / TCR variable and the boundary of the antibody constant / VL domain is aligned with the boundary of CBeta / TCR variable. Examples of drawings of the junction domains are also presented in the form of alignment (see, for example, SEQ ID NO: 172, 173, 174 or 175), where the first or the second junction domain is represented in underlined form. In such embodiments, the first junction domain comprises or is SEQ ID NO: 81, 131, 132 or 133. In such embodiments, the second junction domain comprises or is SEQ ID NO: 49 or 50.

TABLE 4. Drawings of the first and second junction domains for VH-C Pre-Alpha / VL-CBeta Junction Junction (heavy chain) (light chain)

Constant Variable Constant Variable Antibody_H …… LVTVSS --ASTKGPS …… Antibody_L …… KVEIK-- RTVAAPSVF …… SEQ ID NO: 288 SEQ ID NO: 291 TCR-alpha …… KLIIK-- PDIQNPDPA …… TCR_beta …… RLTVLEPDLKNV … SEQ ID NO: 289 SEQ ID NO: 292 Pre-TCR_alpha …… GCPAL-- PTGVGGTPF …… SEQ ID NO: 290 H_Conjunction_C …… LVTVSS ASGVGGTPF …… L_Conjunction_C …… KVEIKLE -DLKNVFPPE …… SEQ ID NO: 172 : 174 H_Conjunction_D …… LVTVSS PTGVGGTPF …… L_Conjunction_D …… KVEIKLE -DLKNVFPPE …… SEQ ID NO: 173 SEQ ID NO: 175

[0274] In certain embodiments, C1 comprises a modified CGama and C2 comprises a modified CDelta. Table 5 illustrates the examples of designs for the junction domains useful for the VCR of antibody fused to TCR gamma, or for the VL of antibody fused to CDelta of TCR. The limit of the antibody constant / VH domain is aligned with the limit of gamma / TCR variable and the limit of the antibody constant / VL domain is aligned with the limit of CDelta / TCR variable. Examples of drawings of the junction domains are also presented in the form of alignment (see, for example, SEQ ID NO: 157, 158, 159 or 160), where the first or the second junction domain is represented in underlined form. In such embodiments, the first junction domain comprises or is SEQ ID NO: 117 or 118. In such embodiments, the second junction domain comprises or is SEQ ID NO: 119 or 120.

TABLE 5. Drawings of the first and second junction domains for for VH-CGama / VL-CDelta Junction Junction (heavy chain) (light chain) Constant Variable Constant Variable Antibody_H …… LVTVSS AS - TK-GPS …… Antibody_L …… KVEIK --- RTVAAPSVF …… SEQ ID NO: 293 SEQ ID NO: 143 TCR_gamma …… TLVVTD KQLDADVSPK …… TCR_delta …… RVTVE PRSQPHTKPSVF …… SEQ ID NO: 142 SEQ ID NO: 55 H_Conjunction_4 …… LVTVSS ASLDADVSPSP… KVEIK PRSQPHTKPSVF …… SEQ ID NO: 157 SEQ ID NO: 159 H_ Conjunction _5 …… LVTVTD KQLDADVSPK …… L_ Conjunction _5 …… KVEIE PRSQPHTKPSVF …… SEQ ID NO: 158 SEQ ID NO: 160

[0275] In certain embodiments, C1 comprises a modified CDelta and C2 comprises a modified CGama. Table 6 illustrates the examples of designs for the junction domains useful for the VH of antibody fused to TCR CDelta, or for the VL of antibody fused to TCR gamma. The limit of the antibody constant / VH domain is aligned with the limit of CDelta / TCR variable and the limit of the antibody constant / VL domain is aligned with the limit of Gamma / TCR variable. Examples of drawings of the junction domains are also presented in the form of alignment (see, for example, SEQ ID NO: 161, 162, 163 or 164), where the first or the second junction domain is represented in underlined form. In such embodiments, the first junction domain comprises or is SEQ ID NO: 123 or 124. In such embodiments, the second junction domain comprises or is SEQ ID NO: 125 or 126.

TABLE 6. Drawings of the first and second junction domains for for VH-CDelta / VL-CGama Junction Junction (heavy chain) (light chain) Constant Variable Constant Variable Antibody_H …… LVTVSS --ASTKGPS …… Antibody_L …… KVEIK- RTV- --AAPSVF …… SEQ ID NO: 56 SEQ ID NO: 58 TCR_delta …… RVTVEP RSQPHTKPS …… TCR_gamma …… TLVVTD KQLDADVSPKPT …… SEQ ID NO: 57 SEQ ID NO: 59 H_Conjunction_6 …… LVTVSS RSQPHTKPS… L… KQLDADVSPKPT …… SEQ ID NO: 161 SEQ ID NO: 163 H_Conjunction_7 …… LVTVEP RSQPHTKPS …… L_Conjunction_7 …… KVEITD KQLDADVSPKPT …… SEQ ID NO: 162 SEQ ID NO: 164

[0276] In certain embodiments, the first polypeptide comprises a sequence comprising domains operably linked as in formula (I): VH-HCJ-C1, and the second polypeptide comprises a sequence comprising domains operably linked as in formula (II): VL-LCJ-C2, where: VH is an antibody heavy chain variable domain; HCJ is a first join domain as defined above; C1 is a first TCR constant domain as defined above; VL is an antibody light chain variable domain; LCJ is a second join domain as defined above; C2 is a second constant domain of TCR as defined above.

[0277] In certain embodiments, C1 is a modified CAlfa that comprises or is a sequence selected from a group consisting of: SEQ ID

NOs: 42-48, and C2 is a modified CBeta that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 51 and 52.

[0278] In certain embodiments, C1 is a modified CBeta that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41 and C2 is a modified CAlfa that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 42-48, the HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 129 and 130; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50.

[0279] In certain embodiments, C1 is a modified CBeta that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, 84, 319, 320, 321, 322, 323 and 324 and C2 is a modified C Pre-Alpha that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317 and 318, HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 81 and

131.

[0280] In certain embodiments, C1 is a modified CPre-Alpha that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 311, 312, 313, 314, 315, 316, 317 and 318 and C2 is a modified CBeta that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 32-41, HCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 81, 131, 132 and 133; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 49 and 50.

[0281] In certain embodiments, C1 is a modified gamma that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 113, 114, 333, 334, 335, 336, 337, 338, 339 and 340, and C2 is a modified Cdelta that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 325, 326, 327, 328, 329, 330, 331 and 332, HCJ comprises or is a selected sequence a group consisting of: SEQ ID NOs: 117 and 118; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 119 and 120.

[0282] In certain embodiments, C1 is a modified CDelta that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 325, 326, 327, 328, 329, 330, 331 and 332, and C2 is a modified gamma that comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 113, 114, 333, 334, 335, 336, 337, 338, 339 and 340, HCJ comprises or is a selected sequence a group consisting of: SEQ ID NOs: 123 and 124; LCJ comprises or is a sequence selected from a group consisting of: SEQ ID NOs: 125 and 126.

[0283] U.S. patent 9,683,052 discloses that certain residues at the contact interface between the regions contained in the TCR can be modified in an Fc region to facilitate the heterodimeric pairing of two heavy chains. Such residues and / or corresponding residues within the contact interface between the regions listed in the TCR disclosed here can also be incorporated into a Fab region, for example, the CH1 and CL domains, to facilitate pairing between a light chain and a heavy chain .

[0284] ii. Antibody variable region

[0285] The polypeptide complex provided here comprises a first heavy chain variable domain (VH) and a first light chain variable domain (VL) of the first antibody. In a conventional native antibody, a variable region comprises three CDR regions interposed by flanking structural (FR) regions, for example, as stated in the following formula: FR1-CDR1-FR2-CDR2- FR3-CDR3-FR4, from the N-terminal end to the C-terminal. The polypeptide complex provided here can comprise, but is not limited to, a conventional antibody variable region. For example, the variable domain can comprise all three or less than three of the CDRs, with all four or less than four of the antibody's heavy or light chain FRs, as long as the variable domain is able to specifically bind to an antigen .

[0286] The first antibody has a first antigen specificity.

In other words, VH and VL form an antigen-binding site that can specifically bind to an antigen or an epitope. The antigenic specificity can be directed to any suitable antigen or epitope, for example, one that is an exogenous antigen, endogenous antigen, autoantigen, neoantigen, viral antigen or tumor antigen. An exogenous antigen enters the body by inhalation, ingestion or injection and can be presented by antigen presenting cells (APCs) by endocytosis or phagocytosis and form the MHC II complex. An endogenous antigen is generated within normal cells as a result of cell metabolism, intracellular viral or bacterial infection, which can form an MHC I complex. An autoantigen (eg, peptides, DNA or RNA, etc.) is recognized by the immune system of a patient suffering from autoimmune diseases, whereas under normal conditions, this antigen should not be the target of the immune system. The neoantigen is completely absent from a normal body and is generated due to a certain disease, such as tumors or cancer. In certain embodiments, the antigen is associated with a particular disease (for example, tumor or cancer, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neuropathies, neuropsychiatric conditions, injuries, inflammations, coagulation disorder). In certain embodiments, the antigen is associated with the immune system (for example, immune cells such as B cells, T cells, NK cells, macrophages, etc.).

[0287] In certain embodiments, the first antigen specificity is directed to an antigen related to the immune system or to an epitope thereof.

Examples of an immune-related antigen include a specific T cell receptor molecule and / or a natural killer cell specific receptor molecule (NK cell).

[0288] The T cell-specific receptor molecule allows a T cell to bind and, if there are additional signals, to be activated and respond to an epitope / antigen presented by another cell called an antigen presenting cell or APC.

The specific T cell receptor molecule can be TCR, CD3, CD28, CD134 (also called OX 0), 4-1BB, CD5 and CD95 (also known as Fas receptor).

[0289] Examples of a specific NK cell receptor molecule include CD16, a low affinity Fc receptor and NKG2D and CD2.

[0290] In certain embodiments, the first antigen specificity is directed to an antigen associated with a tumor or an epitope thereof. The term "tumor-associated antigen" refers to an antigen that is or can be presented on the cell surface of a tumor and that is located on or within the tumor cells. In some embodiments, tumor-associated antigens can be presented only by tumor cells and not by normal, that is, non-tumor cells. In some other embodiments, the tumor-associated antigens can be expressed exclusively in tumor cells or may have a specific tumor mutation in comparison to non-tumor cells. In some other embodiments, tumor-associated antigens can be found in both tumor and non-tumor cells, but are overexpressed in tumor cells when compared to non-tumor cells or are accessible for antibody binding to tumor cells due to less compact structure of tumor tissue compared to non-tumor tissue. In some embodiments, the tumor-associated antigen is located in the vasculature of a tumor.

[0291] Illustrative examples of a tumor-associated surface antigen are CD10, CD19, CD20, CD22, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CD133, Fms-type tyrosine kinase 3 (FLT -3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulphate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, IL3R, fibroblast activating protein (FAP) , CDCP1, Derlin1, Tenascin, frizzled 1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-alpha (CD140a), PDGFR-beta (CD140b) Endoglina, CLEC14, Tem1-8 and Tie2.

Other examples may include A33, CAMPATH-1 (CDw52), carcinoembryonic antigen (CEA), Carboanidrase IX (MN / CA IX), 2-7 EGFR, EGFRvIII, EpCAM, Ep-CAM, folate-binding protein, G250, tyrosine Fms type kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (cell surface chondroitin sulfate proteoglycan associated with melanoma), Muc-1, prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate specific antigen (PSA) and TAG-72.

[0292] In certain embodiments, the first antigen specificity is directed to an antigen or an epitope thereof, selected from the group consisting of: CD3, 4.1BB (CD137), OX40 (CD134), CD16, CD47, CD19, CD20, CD22, CD33, CD38, CD123, CD133, CEA, CDH3, EpCAM, epidermal growth factor receptor (EGFR), EGFRvIII (a mutant form of EGFR), HER2, HER3, dLL3, BCMA, Sialyl-Lea 5T4, ROR1, chondroitin sulfate proteoglycan associated with melanoma, mesothelin, folate receptor 1, VEGF receptor, EpCAM, HER2 / neu,

HER3 / neu, G250, CEA, MAGE, proteoglycans, VEGF, FGFR, alphaVbeta3, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, CD13, CD14, CD21, CD23 , CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, ganglioside GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4 Trp2, gp100, tyrosinase, Muc-1, telomerase, survivin, G250, p53, CA125 MUC, Wue antigen, Lewis Y antigen, HSP-27, HSP -70, HSP-72, HSP-90, Pgp, MCSP, EpHA2 and the cell surface targets GC182, GT468 or GT512.

[0293] The antibody variable domains can be derived from a parent antibody. A parental antibody can be any type of antibody, including, for example, a fully human antibody, a humanized antibody, or an animal antibody (for example, mouse, rat, rabbit, sheep, cow, dog, etc.). The parental antibody can be a monoclonal antibody or a polyclonal antibody.

[0294] In certain embodiments, the parental antibody is a monoclonal antibody. A monoclonal antibody can be produced by various methods known in the art, for example, hybridoma technology, recombinant method, phage display or any combination thereof.

[0295] Hybridoma technology involves fusing B cells that express antibodies with an immortal B cell line to produce hybridomas, which are screened for desirable characteristics, such as high level of antibody production, good growth of hybridoma and strong binding or good biological activity of the antibody (see, for example, Harlow et al., (1988) Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.).

[0296] The recombinant method is another way to produce a parental antibody. Briefly, cells, such as lymphocytes that secrete the antibodies of interest, are obtained and identified and individual cells are isolated, followed by

PCR with reverse transcriptase to produce cDNAs from the heavy and light chain variable region. These variable region cDNA sequences can be used to construct the polypeptide complex coding sequence provided here, and then expressed in a suitable host cell (for review, see, for example, US Patent No. 5,627,052; PCT WO publication 92/02551 and Babcock et al., (1996) Proc. Natl. Acad. Sci. USA 93: 7843-7848).

[0297] Antibody libraries are still an alternative for obtaining a parental antibody. Briefly, an antibody library can be screened to identify an antibody with the desired binding specificity. Methods for such screening of recombinant antibody libraries are well known in the art, and include the methods described, for example, in the U.S.

Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 97/29131; Fuchs et al., (1991) Bio / Technology 9: 1370-1372; Hay et al., (1992) Hum. Antibod.

Hybridomas 3: 81-85; Huse et al., (1989) Science 246: 1275-1281; McCafferty et al., (1990) Nature 348: 552-554; Griffiths et al., (1993) EMBO J. 12: 725-734; Hawkins et al., (1992) J. Mol. Biol. 226: 889-896; Clackson et al., (1991) Nature 352: 624-628; Gram et al., (1992) Proc. Natl. Acad. Sci. USA 89: 3576-3580; Garrad et al., (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al., (1991) Nucl. Acid Res. 19: 4133-4137; and Barbas et al., (1991) Proc. Natl. Acad. Sci. USA 88: 7978-7982; and US Patent Application Publication No. 20030186374.

[0298] Another illustrative method for obtaining a parental antibody is the phage display or phage display (see, for example, Brinkman et al., (1995) J.

Immunol. Methods 182: 41-50 .; Ames et al., (1995) J. Immunol. Methods 184: 177-186; Kettleborough et al., (1994) Eur. J. Immunol. 24: 952-958; Persic et al., (1997) Gene 187 9-18; and US Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;

5,658,727; 5,733,743; and 5,969,108). The polynucleotide sequences encoding the antibody domains are introduced into the phage particles to generate a library of phage particles that exhibit a variety of functional antibody domains. Fd and M13 are commonly used filamentous phages, and the functional antibody domains displayed on the phage particles can be, for example, disulfide stabilized Fab, Fv or Fv antibody domains, which are recombinantly fused to an encoded phage protein by gene III or gene VIII. The phage library can be searched using an antigen of interest, for example, which is optionally labeled and bound or captured on a solid substrate (for example, a sphere). For a selected phage, its polynucleotide sequences that encode the antibody variable domains are obtained and used in the construction of the polypeptide complex provided here. Likewise, a yeast library that exhibits variable antibody domains by attaching antibody domains to the yeast cell wall (see, for example, US Patent No. 6,699,658) can be generated, and then screened with a linked antigen to obtain a parental antibody useful for the construction of the polypeptide complex provided herein.

[0299] In addition, a parental antibody can also be produced by injecting an antigen of interest into a transgenic non-human animal that comprises part or all of the human immunoglobulin locus, for example, OmniRat, OmniMouse (see, for example, Osborn M. et al., Journal of Immunology, 2013, 190: 1481-1490; Ma B. et al., Journal of Immunological Methods 400 - 401 (2013) 78-86; Geurts A. et al., Science, 2009, 325: 433; US patent 8,907,157; EP patent 2152880B1; EP patent 2336329B1), HuMab mice (see, for details, Lonberg, N. et al., Nature 368 (6474): 856 859 (1994)), Xeno-Mouse ( Mendez et al., Nat Genet., 1997, 15: 146-156), TransChromo Mouse (Ishida et al., Cloning Stem Cells, 2002, 4: 91-102) and VelocImmune Mouse (Murphy et al., Proc Natl Acad Sci USA, 2014,

111: 5153-5158), Kymouse (Lee et al., Nat. Biotechnol, 2014, 32: 356–363) and transgenic rabbit (Flisikowska et al., PLoS One, 2011, 6: e21045).

[0300] The parental antibodies described here can be further modified, for example, to engraft the CDR sequences on a different structure or support, to replace one or more amino acid residues in one or more structural regions, to replace one or more residues in one or more CDR regions for affinity maturation, and so on. These can be performed by a person skilled in the art using conventional techniques.

[0301] The parental antibody can also be a therapeutic antibody known in the state of the art, such as those approved by the FDA for therapy or diagnosis, or those undergoing clinical trials to treat a condition, or those undergoing research and development . Polynucleotide and protein sequences for the variable regions of known antibodies can be obtained from public databases, such as, for example, www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html; www.sciquest.com/; www.abcam.com/; www.antibodyresource.com/onlinecomp.html.

[0302] Examples of therapeutic antibodies include, but are not limited to rituximab (Rituxan, IDEC / Genentech / Roche) (see, for example, U.S. Patent No.

5,736,137), a chimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently under development by Genmab, an anti-CD20 antibody described in Pat. US 5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel) and PRO70769 (PCT Application No. PCT / US2003 / 040426), trastuzumab (Herceptin, Genentech) (see, for example, US Pat. 5,667,171), a humanized anti-Her2 / neu antibody approved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg), currently under development by Genentech; an anti-Her2 antibody described in U.S. Patent No.

4,753,894; cetuximab (Erbitux, Imclone) (US Patent No. 4,943,533; PCT Publication

No. WO 96/40210), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers; ABX-EGF (US Patent No. 6,235,883), currently under development by Abgenix-Immunex-Amgen; HuMax-EGFr (US Pat. 7,247,301), currently under development by Genmab; 425, EMD55900, EMD62000 and EMD72000 (Merck KGaA) (US Patent 5,558,864; Murthy et al., (1987) Arch. Biochem.

Biophys. 252 (2): 549-60; Rodeck et al., (1987) J. Cell. Biochem. 35 (4): 315-20; Kettleborough et al., (1991) Protein Eng. 4 (7): 773-83); ICR62 (Institute of Cancer Research) (PCT Publication No. WO 95/20045; Modjtahedi et al., (1993) J. Cell Biophys. 22 (1-3): 129-46; Modjtahedi et al., (1993) Br J. Cancer 67 (2): 247-53; Modjtahedi et al., (1996) Br. J. Cancer 73 (2): 228-35; Modjtahedi et al., (2003) Int. J.

Cancer 105 (2): 273-80); TheraCIM hR3 (YM Biosciences, Canada and Center for Molecular Immunology, Cuba (U.S. Patent No. 5,891,996; U.S. Patent No.

6,506,883; Mateo et al., (1997) Immunotechnol. 3 (1): 71-81); mAb-806 (Ludwig Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al., (2003) Proc.

Natl. Acad. Sci. USA 100 (2): 639-44); KSB-102 (KSBiomedix); MR1-1 (IVAX, National Cancer Institute) (PCT publication No. WO 0162931); and SC100 (Scancell) (PCT WO 01/88138); alemtuzumab (Campath, Millenium), a humanized mAb currently approved for the treatment of chronic B-cell lymphocytic leukemia; muromonab- CD3 (Orthoclone OKT3), an anti-CD3 antibody developed by OrthoBiotech / Johnson & Johnson, ibritumomab tiuxetan (Zevalin), an anti-CD20 antibody developed by IDEC / Schering AG, gemtuzumab ozogamycin (Mylotarg), an anti-CD33 antibody (p67 protein) developed by Celltech / Wyeth, alefacept (Amevive), an anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro), developed by Centocor / Lilly, basiliximab (Simulect), developed by Novartis, palivizumab ( Synagis), developed by Medimmune, infliximab (Remicade), an anti-TNF alpha antibody developed by Centocor, adalimumab (Humira), an anti-TNF alpha antibody developed by Abbott, Humicade, an anti-TNF alpha antibody developed by Celltech, golimumab (CNTO-148), a fully human TNF antibody developed by Centocor, etanercept (Enbrel), a fc p75 TNF receptor fusion developed by Immunex / Amgen, lenercept, a Fc p55TNF receptor fusion previously developed by Roche, ABX- CBL, an anti-CD147 antibody under development by Abgenix, ABX-IL8, an anti-IL8 antibody under development by Abgenix, ABX-MA1, an anti-MUC18 antibody under development by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), an anti-

MUC1 under development by Antisoma, Therex (R1550), an anti-MUC1 antibody under development by Antisoma, AngioMab (AS1405), under development by

Antisoma, HuBC-1, under development by Antisoma, Thioplatin (AS1407) under development by Antisoma, Antegren (natalizumab), an anti-alpha-4- antibody

beta-1 (VLA-4) and alpha-4-beta-7 under development by Biogen, VLA-1 mAb, an anti-integrin antibody VLA-1 under development by Biogen, LTBR mAb, an anti-beta lymphotoxin receptor antibody (LTBR) under development by Biogen,

CAT-152, an anti-TGF-beta 2 antibody under development by Cambridge

Antibody Technology, ABT 874 (J695), an anti-IL-12 p40 antibody under development by Abbott, CAT-192, an anti-TGF beta1 antibody under development by Cambridge Antibody Technology and Genzyme, CAT-213, an anti-Eotaxinl antibody under development by Cambridge Antibody Technology,

LymphoStat-B, an anti-Blys antibody under development by Cambridge Antibody

Technology and Human Genome Sciences Inc., TRAIL-RlmAb, an anti-TRAIL-

R1 in development by Cambridge Antibody Technology and Human Genome

Sciences, Inc., Avastin bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody under development by Genentech, an antibody in the anti-HER receptor family under development by Genentech, an Anti-Tissue Factor (ATF), an anti-

Tissue Factor under development by Genentech, Xolair (Omalizumab), an anti-IgE antibody under development by Genentech, Raptiva (Efalizumab), an anti-CD11a antibody under development by Genentech and Xoma, MLN- Antibody

02 (formerly LDP-02), under development by Genentech and Millenium

Pharmaceuticals, HuMax CD4, an anti-CD4 antibody under development by

Genmab, HuMax-IL15, an anti-IL15 antibody under development by Genmab and

Amgen, HuMax-Inflam, under development by Genmab and Medarex, HuMax-Cancer,

an anti-Heparanase I antibody under development by Genmab and Medarex and

Oxford GcoSciences, HuMax-Lymphoma, under development by Genmab and Amgen,

HuMax-TAC, under development by Genmab, IDEC-131 and anti-CD40L antibody under development by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody under development by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody under development by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 under development by IDEC Pharmaceuticals, anti-macrophage migration factor (MIF) antibodies under development by IDEC Pharmaceuticals,

BEC2, an anti-idiotypic antibody under development by Imclone, IMC-1C11, an anti-KDR antibody under development by Imclone, DC101, an anti-flk-1 antibody under development by Imclone, cadherin anti-VE antibodies under development by Imclone, CEA-Cide (labetuzumab), an antibody to anti-

carcinoembryonic (CEA) under development by Immunomedics, LymphoCide

(Epratuzumab), an anti-CD22 antibody under development by Immunomedics,

AFP-Cide, under development by Immunomedics, MyelomaCide, under development by Immunomedics, LkoCide, under development by

Immunomedics, ProstaCide, under development by Immunomedics, MDX-010, an anti-CTLA4 antibody under development by Medarex, MDX-060, an anti-CD30 antibody under development by Medarex, MDX-070 under development by

Medarex, MDX-018 under development by Medarex, Osidem (IDM-1) and anti-Her2 antibody under development by Medarex and Immuno-Designed Molecules, HuMax-

CD4, an anti-CD4 antibody under development by Medarex and Genmab, HuMax-

IL15, an anti-IL15 antibody under development by Medarex and Genmab, CNTO

148, an anti-TNF alpha antibody. under development by Medarex and Centocor / J & J,

CNTO 1275, an anti-cytokine antibody under development by Centocor / J & J,

MOR101 and MOR102, antibodies to intercellular adhesion molecule-1 (ICAM-1)

(CD54) under development by MorphoSys, MOR201, an anti-fibroblast growth factor receptor (FGFR-3) antibody under development by MorphoSys,

Nuvion (visilizumab), an anti-CD3 antibody under development by Protein Design

Labs, HuZAF, an anti-interferon gamma antibody under development by Protein

Design Labs, Alpha5-beta1 anti-integrin, under development by Protein Design

Labs, anti-IL-12, under development by Protein Design Labs, ING-1, an anti-Ep-CAM antibody under development by Xoma, Xolair (Omalizumab), a humanized anti-IgE antibody developed by Genentech and Novartis and MLN01, an anti-integrin beta2 antibody under development by Xoma.

In another embodiment, therapy includes KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer

Research); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (integrin alfaV.beta.3,

Medimmune); volociximab (integrin alfaV.beta1, Biogen / PDL); human 216 mAb

(glycosylated B cell epitope, NCI); BiTE MT103 (CD19 x CD3 bispecific,

Medimmune); 4G7 x H22 (Bcell.times.FcgammaR1 bispecific, Medarex / Merck KGa);

rM28 (CD28.times.MAPG bispecific, EP Patent No.

EP 1 444 268); MDX447 (EMD

82633) (CD64 ˣ bispecific EGFR, Medarex); Catumaxomab (Removab) (EpCAM ˣ bispecific anti-CD3, Trion / Fres); Ertumaxomab (bispecific HER2 / CD3, Fresenius

Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglina),

Tracon), BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19,

Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20)

(CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20,

Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152)

(CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (receiver CD3 fc, PDL

Biopharma); HeFi-1, CD30, NCI); MDX-060 (CD30, Medarex); MDX-1401 (CD30,

Medarex); SGN-30 (CD30, Seattle Genetics); SGN-33 (Lintuzumab) (CD33, Seattle

Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis);

SGN-40 (CD40, Seattle Genetics); Campathlh (Alemtuzumab) (CD52, Genzyme);

MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38, Immunomedics); Galiximab

(IDEC-144) (CD80, Biogen); MT293 (TRC093 / D93) (cleaved collagen, Tracon);

HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb);

Tremelimumab (Ticilimumab, CP-675.2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab)

(DR4 agonist TRAIL-R1, Human Genome Science / Glaxo Smith Kline); AMG-655

(DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-

ETR2 (lexatumumab) (TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR,

Imclone); IMC-11F8, (EGFR, Imclone); Nimotuzumab (EGFR, YMBio); Panitumumab

(Vetabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110

(EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201) (Epcam, Merck);

edrecolomab (Panorex, 17-1A) (Epcam, Glaxo / Centocor); MORAb-003 (a folate receptor, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9,

Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin)

(HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab

(HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-1R

R1507 (IGF1-R, Roche); CP 751871 (IGF-R, Pfizer); BMI-A12 (IGF1-R, Imclone);

BIIB022 (IGF-1R, Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO

328 (IL6, Centocor); Anti-KIR (1-7F9) (Ig Killer cell receptor (KIR), New);

Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTBR,

Biogen); HuHMFG1 (MUC1, Antisoma / NCI); RAV12 (carbohydrate epitope linked to

N, Raven); CAL (parathyroid hormone-related protein (PTH-rP),

University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1,

Medarex / Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone); bavituximab (phosphatidylserine, Peregrine); hJ591 (PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb inhibitor (pan) (IgG4), Genzyme); Infliximab (Remicade) (TNF alpha, Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO 2005/111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab) PCT Publication No. WO / 2000/034337, University of Texas); IMC-18F1 (VEGFR1, Imclone); IMC-1121 (VEGFR2, Imclone).

a) Anti-CD3 connection portion

[0303] In certain embodiments, the first antigen binding portion or the second antigen binding portion is an anti-CD3 binding portion derived from an anti-CD3 antibody comprising 1, 2 or 3 CDR sequences from heavy chains selected from the group consisting of: SEQ ID NOs: 342-344 and / or 1, 2, or 3 light chain CDR sequences selected from SEQ ID NOs: 345-347.

[0304] These CDR sequences are derived from the anti-CD3 antibody shown in Table A below. The CDR sequences of the WBP3311_2.306.4 antibody are provided below.

Table A Antibody ID: CDR1 CDR2 CDR3 SEQ ID NO: 342 SEQ ID NO: 343 SEQ ID NO: 344

VH WISPGNVNTKYNENF WBP3311_2.306.4 GFAFTDYYIH DGYSLYYFDY

KG SEQ ID NO: 345 SEQ ID NO: 346 SEQ ID NO: 347

VL KSSQSLLNSRTRKN WBP3311_2.306.4 WASTRQS TQSHTLRT

YLA

[0305] The sequences of the variable region of the kappa and heavy chain of the antibody WBP3311_2.306.4 are provided below.

WBP3311_2.306.4-VH Amino acid sequence (SEQ ID NO: 348):

QVQLQQSGPELVKPGASVRISCKASGFAFTDYYIHWVKQRPGQGLEWIGWISPGNVNTKYNE

NFKGRATLTADLSSSTAYMQLSSLTSEDSAVYFCARDGYSLYYFDYWGQGTTLTVSS Nucleic acid sequence (SEQ ID NO: 349):

CAGGTCCAGCTGCAGCAGTCTGGACCTGAATTGGTGAAGCCTGGGGCTTCCGTGAGGATATC CTGCAAGGCTTCTGGCTTCGCCTTCACAGACTACTATATACACTGGGTTAGAGCAGAGGCCTG GACAGGGTCTTGAGTGGATTGGATGGATTTCTCCTGGAAATGTTAATACTAAATACAATGAA AACTTCAAGGGCAGGGCCACACTGACTGCAGACCTATCCTCCAGCACAGCCTACATGCAGCT CAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGATGGATATTCCCTGT

ATTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA WBP3311_2.306.4-V L Amino acid sequence (SEQ ID NO: 350):

DIVMSQSPSSLTVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRQS

GVPDRFTGSGSGTAFTLTISGVQAEDLAVYFCTQSHTLRTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 351):

GACATTGTGATGTCACAGTCTCCATCCTCCCTGACTGTGTCAGCAGGAGAGAAGGTCACTAT GAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGT ACCAGCAGAAGCCAGGGCAGTCTCCTAAACTACTAATCTACTGGGCATCCACTAGGCAATCT GGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGCTTTCACTCTCACCATCAGCGG TGTGCAGGCTGAAGACCTGGCAGTTTATTTCTGCACGCAATCTCATACTCTTCGGACGTTCG GTGGAGGCACCAAGCTGGAAATCAAA

[0306] CDRs are known to be responsible for binding to the antigen, however, it has been found that not all 6 CDRs are indispensable or immutable. In other words, it is possible to replace or alter or modify one or more CDRs in the anti-CD3 binding portion derived from WBP3311_2.306.4, while still maintaining substantially the specific binding affinity for CD3.

[0307] In certain embodiments, the anti-CD3 binding portion provided herein comprises a heavy chain CDR3 sequence from one of the anti-CD3 antibodies WBP3311_2.306.4. In certain embodiments, the anti-CD3 binding portion provided herein comprises a heavy chain CDR3 comprising SEQ ID NO. 344. The heavy chain CDR3 regions are located at the center of the antigen-binding site and therefore are believed to make the greatest contact with the antigen and provide more free energy to the antibody's affinity for the antigen. It is also believed that heavy chain CDR3 is by far the most diverse CDR of the antigen binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S., Nature. 302: 575-81 (1983)). The diversity in the CDR3 of the heavy chain is sufficient to produce most of the specificities of the antibodies (Xu JL, Davis MM., Immunity. 13: 37-45 (2000)), as well as the desirable antigen binding affinity (Schier R, et al., J Mol Biol. 263: 551-67 (1996)).

[0308] In certain embodiments, the anti-CD3 binding portion provided herein comprises the appropriate structural region (FR) sequences, provided that the anti-CD3 binding portion can specifically bind to CD3. The CDR sequences provided in Table A are obtained from mouse antibodies, but can be grafted into any suitable FR sequences of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the state of art, such as recombinant techniques.

[0309] In certain embodiments, the anti-CD3 binding portion provided here is humanized.

[0310] In certain embodiments, the humanized antigen binding portion provided here is composed substantially of all human sequences, except for non-human CDR sequences. In some embodiments, the FRs variable region and constant regions, if present, are derived wholly or substantially from human immunoglobulin sequences. Human FR sequences and human constant region sequences can be derived from different human immunoglobulin genes, for example, FR sequences derived from a human antibody and constant region from another human antibody. In some embodiments, the humanized antigen-binding portion comprises human FR1-4.

[0311] The sequences of the variable region of the heavy chain and the light chain of the humanized anti-CD3 antibody, WBP3311_2.306.4-z1, are provided below.

WBP3311_2.306.4-z1-VH Amino acid sequence (SEQ ID NO: 352):

QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWISPGNVNTKYNE

NFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYYFDYWGQGTLVTVSS Nucleic acid sequence (SEQ ID NO: 353):

CAGGTGCAGCTTGTGCAGTCTGGGGCAGAAGTGAAGAAGCCTGGGTCTAGTGTCAAGGTGTC ATGCAAGGCTAGCGGGTTCGCCTTTACTGACTACTACATCCACTGGGTGCGGCAGGCTCCCG GACAAGGGTTGGAGTGGATGGGATGGATCTCCCCAGGCAATGTCAACACAAAGTACAACGAG AACTTCAAAGGCCGCGTCACCATTACCGCCGACAAGAGCACCTCCACAGCCTACATGGAGCT GTCCAGCCTCAGAAGCGAGGACACTGCCGTCTACTACTGTGCCAGGGATGGGTACTCCCTGT

ATTACTTTGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC WBP3311_2.306.4-z1-VL Amino acid sequence (SEQ ID NO: 354):

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRQS

GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRTFGGGTKVEIK Nucleic acid sequence (SEQ ID NO: 355):

GATATCGTGATGACCCAGAGCCCAGACTCCCTTGCTGTCTCCCTCGGCGAAAGAGCAACCAT CAACTGCAAGAGCTCCCAAAGCCTGCTGAACTCCAGGACCAGGAAGAATTACCTGGCCTGGT ATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTCATCTACTGGGCCTCCACCCGGCAGTCT GGGGTGCCCGATCGGTTTAGTGGATCTGGGAGCGGGACAGACTTCACATTGACAATTAGCTC ACTGCAGGCCGAGGACGTGGCCGTCTACTACTGTACTCAGAGCCACACTCTCCGCACATTCG

GCGGAGGGACTAAAGTGGAGATTAAG 1b) Anti-CD19 antibody

[0312] In certain embodiments, the first antigen binding portion or the second antigen binding portion is an anti-CD19 binding portion derived from an anti-CD19 antibody comprising 1, 2 or 3 chain CDR sequences heavy selected from the group consisting of SEQ ID NOs: 356-359 and / or kappa light chain CDR sequences of 1, 2 or 3 selected from the group consisting of: SEQ ID NOs: 360-362.

[0313] These CDR sequences are derived from the antibodies shown in Table B below. The CDR sequences of the anti-CD19 antibodies are provided below.

Table B CDR1 CDR2 CDR3 SEQ ID NO: 356 SEQ ID NO: 357 SEQ ID NO: 358 WBP7011_4.155.8 VH YIDPYNGDTTYNQKF

GYAFTSYNMY TAYAMDY KG

SEQ ID NO: 356 SEQ ID NO: 359 SEQ ID NO: 358 W7011-4.155.8-z1-

VH YIDPYNADTTYNQKF P15 GYAFTSYNMY TAYAMDY

KG SEQ ID NO: 360 SEQ ID NO: 361 SEQ ID NO: 362 WBP7011_4.155.8 VL

SASSTVNYMH STSNLAS HQWSSYPYT W7011-4.155.8-z1- SEQ ID NO: 360 SEQ ID NO: 361 SEQ ID NO: 362

VL P15 SASSTVNYMH STSNLAS HQWSSYPYT

[0314] The sequences of the variable region of the kappa and heavy chain of the antibody WBP7011_4.155.8 are provided below.

WBP7011-4.155.8-VH Amino acid sequence (SEQ ID NO: 363):

EIQLQQSGPELVKPGASVKVSCKASGYAFTSYNMYWVKQSHGKSLEWIGYIDPYNGDTTYNQ

KFKGKATLTVDKSSSTAYMHLNSLTSEDSAVYYCLTTAYAMDYWGQGTSVTVSS Nucleic acid sequence (SEQ ID NO: 364):

GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGGTATC CTGCAAGGCTTCTGGTTATGCATTCACTAGCTACAACATGTACTGGGTGAAGCAGAGCCATG GAAAGAGCCTTGAGTGGATTGGATATATTGATCCTTACAATGGTGATACTACCTACAACCAG AAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGCATCT CAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTCTCACTACGGCCTATGCTATGG

ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA WBP7011-4.155.8-VL Amino acid sequence (SEQ ID NO: 365):

QIVLTQSPAIMSASLGEEITLTCSASSTVNYMHWYQQKSGTSPKLLIYSTSNLASGVPSRFS

GSGSGTFYSLTIRSVEAEDAADYYCHQWSSYPYTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 366):

CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGGAGATCACCCT AACCTGCAGTGCCAGCTCGACTGTAAATTACATGCACTGGTACCAGCAGAAGTCAGGCACTT CTCCCAAACTCTTGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTTCTCGCTTCAGT GGCAGTGGGTCTGGGACCTTTTATTCTCTCACAATCAGAAGTGTGGAGGCTGAAGATGCTGC CGATTATTACTGCCATCAGTGGAGTAGTTATCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAA

[0315] In certain embodiments, the anti-CD19 antibody binding portion described herein comprises a heavy chain CDR3 sequence of the anti-CD19 antibody, WBP7011_4.155.8 or W7011-4.155.8-Z1-P15. In certain embodiments, the anti-CD19 antibody binding portion provided herein comprises a heavy chain CDR3 sequence comprising SEQ ID NO: 358. The heavy chain CDR3 regions are located at the center of the antigen binding site and therefore, it is believed that it makes the greatest contact with the antigen and provides more free energy to the antibody's affinity for the antigen. It is also believed that heavy chain CDR3 is by far the most diverse CDR of the antigen binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S., Nature. 302: 575-81 (1983)). The diversity in the CDR3 of the heavy chain is sufficient to produce most of the specificities of the antibodies (Xu JL, Davis MM., Immunity. 13: 37-45 (2000)), as well as the desirable antigen binding affinity (Schier R, et al., J Mol Biol. 263: 551-67 (1996)).

[0316] In certain embodiments, the anti-CD19 antibodies disclosed here are humanized. The sequences of the light chain and heavy chain variable region for the humanized anti-CD19 antibody, W7011-4.155.8-Z1-P15, are provided below.

W7011-4.155.8-z1-P15-VH Amino acid sequence (SEQ ID NO: 367):

QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLEWIGYIDPYNADTTYNQ

KFKGRVTITRDMSTSTAYMELSSLRSEDTAVYYCLTTAYAMDYWGQGTLVTVSS Nucleic acid sequence (SEQ ID NO: 368):

CAAATGCAGCTCGTCCAGTCTGGACCTGAAGTGAAGAAGCCCGGGACATCCGTCAAGGTCTC ATGTAAGGCTAGCGGGTACGCATTCACTTCCTACAACATGTACTGGGTGCGCCAGGCCAGAG GACAGAGGTTGGAGTGGATCGGCTACATCGACCCATACAACGCCGATACTACCTACAATCAG AAGTTTAAAGGGCGGGTGACCATTACCCGGGATATGTCCACCTCCACCGCCTACATGGAGCT GAGCAGCCTGAGGAGCGAGGACACAGCCGTGTACTACTGCCTGACAACAGCCTATGCCATGG

ACTATTGGGGCCAGGGCACACTTGTGACTGTGAGCAGT W7011-4.155.8-z1-P15-VL Amino acid sequence (SEQ ID NO: 369):

DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAPKLLIYSTSNLASGVPSRFS

GSGSGTEFTLTISSLQPEDFATYYCHQWSSYPYTFGQGTKLEIK Nucleic acid sequence (SEQ ID NO: 370):

GACATCCAGCTCACCCAATCCCCTTCTTTCCTCTCCGCAAGTGTCGGAGATAGGGTGACTAT CACCTGCTCAGCTTCTTCAACCGTGAACTACATGCATTGGTACCAGCAGAAGCCCGGGAAAG CCCCAAAGCTGCTGATCTACAGCACCTCCAATCTGGCCAGTGGAGTGCCAAGCCGGTTTAGC GGGAGCGGCTCCGGCACTGAATTCACTTTGACAATTAGCAGCCTTCAGCCTGAGGACTTTGC CACATATTACTGTCACCAGTGGTCCAGCTACCCCTACACATTCGGGCAGGGCACAAAGCTGG

AGATTAAG Bispecific polypeptide complexes

[0317] In one aspect, the present disclosure here provides a bispecific polypeptide complex. The term "bispecific", as used in this document, means that there are two antigen-binding portions, each of which is capable of binding specifically to a different antigen or to a different epitope on the same antigen. The bispecific polypeptide complex provided here comprises a first antigen binding portion comprising a first heavy chain variable domain operably linked to a first constant region (C1) of the TCR and a first light chain variable domain operably linked to a second constant region ( C2) of the TCR, wherein C1 and C2 are capable of forming a dimer comprising at least one non-native, stabilizing interchain bond between C1 and C2. The bispecific polypeptide complex provided herein further comprises a second antigen-binding portion comprising a second antigen-binding site, but does not contain a sequence derived from a TCR constant region.

[0318] In certain embodiments, the present disclosure provides a bispecific polypeptide complex, comprising a first antigen-binding portion associated with a second antigen-binding portion, wherein: the first antigen-binding portion comprising: a first polypeptide comprising, from the N-terminus to the C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1), and a second polypeptide comprising, from the N-terminal to the C-terminal end, a first light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR,

where: C1 and C2 are capable of forming a dimer comprising at least one non-native interlinking between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interlinking is able to stabilize the dimer, and the first antibody has a first antigen specificity, a second antigen-binding portion has a second antigen specificity that is different from the first antigen specificity, and the first antigen-binding portion and the second antigen-binding portion are less prone to incorrect pairing of what it would be otherwise if both the first and second antigen binding portions were equivalent to the natural Fab.

[0319] The bispecific polypeptide complex provided here is significantly less likely to have heavy and light chain variable domains with incorrect pairing. Without being linked to any theory, it is believed that the TCR constant regions stabilized in the first antigen-binding portion can specifically associate and therefore contribute to the highly specific matching of the intended VH1 and VL1, discouraging unwanted VH1 mismatches or VL1 with other variable regions that do not have the desired antigen binding sites.

[0320] Bispecific polypeptide complexes in WuXiBody formats have a longer in vivo half-life and are relatively easier to manufacture when purchased with bispecific polypeptide complexes in other formats.

[0321] In certain embodiments, the second bispecific polypeptide complex antigen binding portion provided herein comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2) of a second antibody. In certain embodiments, at least one of VH2 and VL2 is operably linked to an antibody constant region, or both VH2 and VL2 are operably linked to antibody heavy chain and light chain constant regions, respectively. In certain embodiments, the second antigen-binding portion further comprises a CH1 constant domain of the antibody operably linked to VH2, and a light chain constant domain of the antibody operably linked to VL2. For example, the second antigen-binding portion comprises a Fab.

[0322] Where a first, second, third and fourth variable domains (for example, VH1, VH2, VL1 and VL2) are expressed in a cell, it is highly desirable that VH1 specifically parallels VL1 and VH2 specifically parallels VL2 so that the resulting bispecific protein product has the correct antigen binding specificities. However, in existing technologies, such as hybrid hybridoma (or quadroma), the random pairing of VH1, VH2, VL1 and VL2 occurs and, therefore, results in the generation of up to ten different species, of which only one is the binding to bispecific functional antigen. This not only reduces the production yield, but also complicates the purification of the target product.

[0323] The bispecific polypeptide complexes provided here are exceptional in that the variable domains are less likely to mismatch than would otherwise be the case if both the first and second antigen-binding portions were equivalent to the natural Fab. In an illustrative example, the first antigen-binding domain comprises VH1-C1 paired with VL1-C2, and the second antigen-binding domain comprises VH2-CH1 paired with VL2-CL. Surprisingly, it was found that C1 and C2 preferentially associate with each other and are less likely to associate with CL or CH1, thus the formation of unwanted pairs such as C1-CH, C1-CL, C2-CH and C2-CL is discouraged and significantly reduced. As a result of the specific association of C1-C2, VH1 specifically pairs with VL1, thus producing the first antigen-binding site, and CH1 specifically pairs with CL, thereby allowing specific VH2- VL2 that provides the second antigen-binding site. Therefore, the first antigen binding portion and the second antigen binding portion are less prone to mismatch, and the mismatches between, for example, VH1-VL2, VH2-VL1, VH1- VH2, VL1-VL2 would be significantly reduced, than it would be otherwise if both the first and second antigen binding portions were equivalent to the natural Fab, for example, in the form of VH1-CH1, VL1-CL, VH2-CH1 and VL2-CL.

[0324] In certain embodiments, the bispecific polypeptide complex provided here, when expressed by a cell, would have significantly fewer products with mismatches (for example, at least 1, 2, 3, 4, 5 or more products with mismatches ) and / or significantly higher production yield (eg at least 10%, 20%, 30%, 40%, 50%, 60% or much higher yield) than a reference molecule expressed under comparable conditions, where the reference molecule is somewhat identical to the bispecific polypeptide complex, except that it has a native CH1 in place of C1 and a native CL in place of C2.

[0325] In certain embodiments, the first and / or the second antigen-binding portion is multivalent, such as bivalent, trivalent, tetravalent. The term "brave", as used in this document, refers to the presence of a specific number of antigen-binding sites in a given molecule. As such, the terms "bivalent", "tetravalent" and "hexavalent" denote the presence of two binding sites, four binding sites and six binding sites, respectively, in an antigen-binding molecule. A bivalent molecule can be monospecific if the two binding sites are both for specific binding of the same antigen or epitope. Likewise, a trivalent molecule can be bispecific,

for example, when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope). In certain embodiments, the first and / or the second antigen binding portion of the bispecific polypeptide complex provided herein can be bivalent, trivalent or tetravalent, with at least two specific binding sites for the same antigen or epitope. This, in certain embodiments, provides a stronger bond to the antigen or epitope than a monovalent equivalent. In certain embodiments, in a bivalent antigen binding portion, the first binding site valence and the second binding site valence are structurally identical (i.e., having the same sequences) or structurally different (i.e., having different sequences, albeit with the same specificity).

[0326] In certain embodiments, the first and / or the second antigen-binding portion is multivalent and comprises two or more antigen-binding sites operably linked together, with or without a spacer.

[0327] In certain embodiments, the first and / or second antigen-binding portion comprises one or more Fab, Fab ', Fab'-SH, F (ab') 2, Fd, Fv and scFv fragments and others fragments described in Spiess et al., 2015, supra and Brinkmann et al., 2017, supra, or the combination thereof, which are connected with or without a spacer in the heavy chain and / or in the light chain and form at least one capable binding to a second antibody.

[0328] In certain embodiments, the second antigen-binding portion comprises two or more Fabs of the second antibody. The two Fabs can be operationally linked to each other, for example, the first Fab can be covalently linked to the second Fab via heavy chain, with or without a spacer between them.

[0329] In certain embodiments, the first antigen-binding portion further comprises a first dimerization domain and the second antigen-binding portion further comprises a second dimerization domain. The term "dimerization domain", as used in this document, refers to the peptide domain that is able to associate to form a dimer, or in some examples, allows spontaneous dimerization of two peptides.

[0330] In certain embodiments, the first dimerization domain can be associated with the second dimerization domain. The association can be through any interaction or connection or suitable connection, for example, through a connector, a disulfide bond, a hydrogen bond, electrostatic interaction, a salt bridge or hydrophobic-hydrophilic interaction, or a combination of them . Examples of dimerization domains include, without limitation, the antibody hinge region, an antibody CH2 domain, an antibody CH3 domain, and other suitable protein monomers capable of dimerization and associating with one another. The hinge region, the CH2 and / or CH3 domain can be derived from any antibody isotype, such as IgG1, IgG2 and IgG4.

[0331] In certain embodiments, the first and / or the second dimerization domains comprise at least a portion of an antibody hinge region. In certain embodiments, the first and / or the second dimerization domains may further comprise an antibody CH2 domain and / or an antibody CH3 domain. In certain embodiments, the first and / or the second dimerization domains comprise at least a portion of the hinge-Fc region, i.e., hinge-CH2-CH3 domain. In certain embodiments, the first dimerization domain can be operationally linked to the C-terminal end of the first constant region of the TCR. In certain embodiments, the second dimerization domain can be operationally linked to the C-

terminal of the antibody CH1 constant region of the second antigen-binding portion.

[0332] In certain embodiments, the first dimerization domain is operationally linked to (with or without a spacer between) the first constant region (C1) of the TCR in a third junction domain.

[0333] If the Fv region of an immunoglobulin is aligned with an immunoglobulin-like TCR domain, the N-terminal of the antibody hinge and the N-terminal of the TCR hinge would also be aligned. An example is given in Table 7 below, where the N-terminal of the antibody hinge (SEQ ID NO: 278 or 279) is aligned with the N-terminal of the hinge of the TCR Beta (SEQ ID NO: 280).

[0334] The third junction domain of the bispecific polypeptide complex, provided here, can be selected in such a way that it comprises an appropriate length (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the N-terminal of the antibody hinge and an appropriate length (for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) of the N-terminal of the TCR hinge. The term "hinge N-terminus", as used in this document, refers to the most N fragment of the hinge region. For example, the join domain can be selected to have all, or most, or some strings of the N-terminal of the antibody hinge or N-terminal of the TCR hinge, or may comprise more residues of the N-terminal of the antibody hinge than of the N-terminal of the TCR hinge, or vice versa.

[0335] In certain embodiments, the third junction domains of the polypeptide complex, provided here, have a total length comparable to that of the N-terminal of the antibody hinge or the N-terminal of the TCR hinge.

[0336] Likewise, a suitable third join domain can be determined on a structural basis. For example, the three-dimensional structures of the antibody and TCR can be overlapped and the overlapping of the N-terminal of the antibody hinge and the N-terminal of the TCR hinge on the overlapping structure can be determined and considered when determining the length or ratio of the N-terminal sequences of the antibody or TCR hinge.

[0337] In certain embodiments, the third junction domain comprises a spacer between the fragments of the N-terminal of the antibody hinge and the N-terminal of the TCR hinge. Any suitable sequences or the length of the spacer sequences can be used, as long as it does not negatively affect the antigen binding or the stability of the polypeptide complex.

[0338] Examples of sequences from the N-terminal of the antibody hinge, the N-terminal of the TCR hinge and the third junction domain are provided in Tables 7, 8, 9 and 10 below.

[0339] In certain embodiments, C1 comprises a modified CBeta and the first dimerization domain comprises the hinge and Fc of IgG1 or IgG4. Table 7 illustrates the examples of designs for the junction domains useful for the TCR CBeta fused to the antibody hinge. The N-terminal of the antibody hinge is aligned with the N-terminal of the hinge of the TCR Beta.

Examples of designs of the junction domains are also presented in the form of alignment (see, for example, SEQ ID NO: 152 or 153). In such embodiments, the third junction domain is comprised in SEQ ID NO: 53 or 54 (which covers the third junction domain and the hinge C-terminal).

TABLE 7. Drawings of the third junction domain for VH-CBeta- Hinge SEQ ID NO IgG1_Antibody_H EPKS-CDKTHTC …… 278 IgG4_Antibody_H ESK ---- YGPPC …… 279 TCR_beta WGRADCGFTSVS …… 280 Conjunction'_1 (SDGTC1) …… 152 Conjunction'_1 (IgG4) WGR ---- YGPPC …… 153

[0340] In certain embodiments, C1 comprises a modified CAlfa or CPré-Alpha and the first dimerization domain comprises hinge and IgG1 or IgG4 Fc. Table 8 illustrates the examples of designs for the junction domains useful for CAlfa or CPré-Alpha from TCR fused to the antibody hinge. The N-terminal of the antibody hinge is aligned with the N-terminal of the Hinge of Alpha or C Pre-Alpha of the TCR. In such embodiments, the third junction domain is comprised in SEQ ID NO: 134, 135, 140 or 141 (which encompasses the third junction domain and the hinge C-terminal).

TABLE 8. Drawings of the third junction domain for VH-CAlfa- Hinge SEQ ID NO IgG1_Antibody_H EPKS-CDKTHTC …… 281 IgG4_Antibody_H ESK ---- YGPPC …… 282 TCR_alpha or TCR_pre-alpha --------- --- Conjunction'_2 (IgG1) ----- SDKTHTC …… 154 Conjunction'_2 (IgG4) ------- YGPPC …… 155

[0341] In certain embodiments, C1 comprises a modified gamma and the first dimerization domain comprises hinge and IgG1 or IgG4 Fc. Table 9 illustrates the examples of designs for the junction domains useful for the TCR gamma fused to the antibody hinge. The N-terminal of the antibody hinge is aligned with the N-terminal of the Hinge of the TCR Gamma.

Examples of designs of the junction domains are also presented in the form of alignment (see, for example, SEQ ID NO: 165 or 166). In such embodiments, the third junction domain is comprised in SEQ ID NO: 121 or 122 (which covers the third junction domain and the hinge C-terminal).

TABLE 9. Drawings of the third junction domain for VH-CGama- Hinge SEQ ID NO IgG1_Antibody_H EPKSCDKTHTC …… 60 IgG4_Antibody_H ESK --- YGPPC …… 61 TCR_gamma PPIKTDVITMD …… 62

Conjunction’_3 (IgG1) PPIKSDKTHTC …… 165 Conjunction’_3 (IgG4) PPI --- YGPPC …… 166

[0342] In certain embodiments, C1 comprises a modified CDelta and the first dimerization domain comprises hinge and Fc of IgG1 or IgG4. Table 10 illustrates the examples of designs for the junction domains useful for the TCR CDelta fused to the antibody hinge. The N-terminal of the antibody hinge is aligned with the N-terminal of the hinge of the Delta TCR.

Examples of joining domain designs are also presented in the form of alignment. In such embodiments, the third junction domain is comprised in SEQ ID NO: 127 or 128 (which covers the third junction domain and the hinge C-terminal).

TABLE 10. Drawings of the third junction domain for VH-CDelta-hinge-Fc Hinge and Fc SEQ ID NO IgG1_Antibody_H EPKSCDKTHTC …… 63 IgG4_Antibody_H ESK --- YGPPC …… 103 TCR_delta FEVKTDSTDHV …… 104 Conjunction_4 … 167 Conjunction'_4 (IgG4) ESK --- YGPPC …… 168

[0343] In certain embodiments, the first dimerization domain is operationally linked to the C-terminal end of a modified TCR constant region and, together, forms a chimeric constant region. In other words, the chimeric constant region comprises the first dimerization domain operationally linked to the modified TCR constant region.

[0344] In certain embodiments, the chimeric constant region comprises a modified CBeta linked to the first hinge-Fc region derived from IgG1, IgG2 or IgG4. Examples of sequences from such a chimeric constant region are provided in Tables 11, 12, 13 and 14.

[0345] In certain embodiments, the chimeric constant region comprises a modified CAlfa attached to the first hinge derived from IgG1,

IgG2 or IgG4. Examples of sequences from such a chimeric constant region are provided in Tables 11, 12 and 13.

[0346] In certain embodiments, the chimeric constant region comprises a modified CPré-Alpha linked to the first hinge derived from IgG1, IgG2 or IgG4, in the third junction domain that comprises or is SEQ ID NO: 134, 135, 140 or 141. Examples of sequences from such a chimeric constant region are provided in Tables 15 and 16.

[0347] In certain embodiments, the chimeric constant region comprises a modified gamma attached to the first hinge derived from IgG1, IgG2 or IgG4. Examples of sequences from such a chimeric constant region are provided in Tables 17 and 18.

[0348] In certain embodiments, the chimeric constant region comprises a modified CDelta attached to the first hinge derived from IgG1, IgG2 or IgG4. Examples of sequences from such a chimeric constant region are provided in Tables 17 and 18.

[0349] In certain embodiments, the chimeric constant region further comprises a first antibody CH2 domain, and / or a first antibody CH3 domain. For example, the chimeric constant region further comprises a first antibody CH2-CH3 domain linked to the C-terminal end of the third junction domain. Examples of sequences from such a chimeric constant region are provided in Table 19.

[0350] In certain embodiments, the first chimeric constant region and the second TCR constant domain comprise a pair of sequences selected from the group consisting of SEQ ID NOs: 177/176, 179/178, 184/183, 185 / 183, 180/176, 181/178, 182/178, 184/186, 185/186, 188/187, 196/187, 190/189, 192/191, 192/193, 195/194, 198/197, 200/199, 202/201, 203/201, 203/204, 205/204, 206/204, 208/207, 208/209, 211/210, 213/212, 213/151, 214/212, 214 / 151, 234/233,

232/231, 216/215, 218/217, 220/219, 222/221, 224/223, 226/225, 227/223, 229/228, 229/230, 236/235 and 238/237, as shown in Table 19.

[0351] These pairs of chimeric constant regions and second TCR constant domains are useful in that they can be modified to fuse with a desired antibody variable region, to provide the polypeptide complex, as disclosed herein. For example, a variable region of the antibody heavy chain can be fused to the chimeric constant region (comprising C1), thereby producing the first polypeptide chain of the polypeptide complex provided here; and similarly, an antibody light chain variable region can be fused to the second constant domain of the TCR (comprising C2), thereby producing the second polypeptide chain of the polypeptide complex provided herein.

[0352] These pairs of chimeric constant regions and second TCR constant domains can be used as a platform to generate the first antigen-binding portion of the bispecific polypeptide complexes provided here. For example, the variable regions of a first antibody can be fused at the N-terminal end of the platform sequences (for example, fusing VH to the chimeric constant domain and VL to the TCR constant domain, respectively). To produce the bispecific polypeptide complex, the second antigen-binding portion can be modified and produced, in order to associate with the bispecific polypeptide complex provided here.

[0353] In certain embodiments, the second dimerization domain comprises a hinge region. The hinge region can be derived from an antibody, such as IgG1, IgG2 or IgG4. In certain embodiments, the second dimerization domain can optionally further comprise an antibody CH2 domain and / or an antibody CH3 domain, for example, as a hinge Fc region. The hinge region can be attached to the antibody heavy chain of the second antigen binding site (e.g., Fab).

[0354] In the bispecific polypeptide complex, the first and second dimerization domains are able to associate with a dimer. In certain embodiments, the first and second dimerization domains are different and are associated in a way that discourages homodimerization and / or favors heterodimerization. For example, the first and second dimerization domains can be selected so that they are not identical and that they preferably form heterodimers with each other, rather than forming homodimers themselves. In certain embodiments, the first and second dimerization domains are able to associate with heterodimers through the formation of knob-into-holes, hydrophobic interaction, electrostatic interaction, hydrophilic interaction or greater flexibility.

[0355] In certain embodiments, the first and second dimerization domains comprise CH2 and / or CH3 domains which are respectively mutated to be able to form a knob-into-holes. A knob can be obtained by replacing a small amino acid residue with a larger one in the first CH2 / CH3 polypeptide, and a hole can be obtained by replacing a large residue with a smaller one. For details of the mutation sites for knob-into-holes, see Ridgway et al., 1996, supra, Spiess et al., 2015, supra and Brinkmann et al., 2017, supra.

[0356] In certain embodiments, the first and second dimerization domains comprise a first CH3 domain of the IgG1 isotype containing the substitutions S139C and T151W (SEQ ID NO: 295, knob) and a second CH3 domain of the IgG1 isotype containing the substitutions Y134C, T151S, L153A and Y192V (SEQ ID NO: 296, hole). In other embodiments, the first and second dimerization domains comprise a first CH3 domain of the IgG4 isotype containing the substitutions S136C and T148W (SEQ ID NO: 298, knob) and a second CH3 domain of the IgG4 isotype containing the substitutions Y131C, T148S, L150A and Y189V (SEQ ID NO: 299, hole). The IgG1 Fc (SEQ ID NO: 294) and IgG4 (SEQ ID NO: 297) Fc sequences and numbering are shown in Figures 20A-20D.

As noted above, XnY when referring to the Fc region (for example, CH3 domain of the Fc region), the numbering of the amino acid residue is based on the numbering shown in Figures 20A-20D.

[0357] In certain embodiments, the first and second dimerization domains further comprise a first hinge region and a second hinge region. For example, replacement load pairs can be introduced into the hinge region of IgG1 and IgG2 to promote heterodimerization. For details, see Brinkmann et al., 2017, supra.

Bispecific format

[0358] The bispecific polypeptide complex provided herein can be in any suitable bispecific format known in the art. In certain embodiments, the bispecific polypeptide complex is based on a bispecific antibody reference format. “Based on,” as used in this document in relation to a bispecific format, means that the bispecific polypeptide complex provided here assumes the same bispecific format as a reference bispecific antibody, except that one of the antigen binding portions has been modified to understand a VH operatively linked to C1 and a VL operably linked to C2, where C1 and C2 are associated with at least one non-native interchain link, as defined above. Examples of bispecific reference antibody formats known in the art include, without limitation, (i) a bispecific antibody with symmetric Fc, (ii) a bispecific antibody with asymmetric Fc, (iii) a regular antibody attached to a portion of additional antigen binding, (iv) a bispecific antibody fragment, (v) a regular antibody fragment attached with an additional antigen binding portion, (vi) a bispecific antibody attached to human albumin or human albumin-binding peptide.

[0359] BsIgG is monovalent for each antigen and can be produced by coexpressing two light and two heavy chains in a single host cell.

An attached IgG is modified to form a bispecific IgG by attaching the terminal amino or carboxyl ends of the light or heavy chains with additional antigen-binding units. Additional antigen-binding units can be single domain antibodies (unpaired VL or VH), such as DVD-Ig, paired antibody variable domains (for example, Fv or scFv) or modified protein structures. Any of the antigen binding units in BsIgG, in particular, paired VH-CH1 / VL-CL, can be modified to replace CH1 with C1 and CL with C2, as disclosed herein, to produce the bispecific polypeptide complex, as provided on here.

[0360] Non-bispecific antibody fragments are antigen-binding fragments that are derived from an antibody, but lack some or all of the antibody constant domains. Examples of such a bispecific antibody fragment include, for example, single domain antibody, Fv, Fab and diabody.

To produce the bispecific polypeptide complex, as provided herein, an antigen binding site (for example, VH-CH1 / VL-CL) on a bispecific antibody fragment, can be modified to understand the polypeptide complex, as disclosed herein (for example, example, VH-C1 / CL-C2).

[0361] In certain embodiments, the bispecific polypeptide complex, as provided herein, is based on the shape of a "complete" antibody, such as complete IgG or IgG-like molecules, and small recombinant formats, such as molecules of tandem single-chain variable fragment (taFvs), diabodies (Dbs), single-chain diabodies (scDbs) and various other derivatives thereof (according to the formats of bispecific antibodies, as described by Byrne H. et al. (2013) Trends Biotech, 31 (11): 621-632. Examples of bispecific antibodies are based on a format that includes, but is not limited to, the chemically coupled Fab (antigen binding fragment), eBiTE (Bispecific T cell engager).

[0362] In certain embodiments, the bispecific polypeptide complex, as provided herein, is based on a bispecific format selected from Triomabs; hybrid hybridoma (quadroma); multispecific platform anticalin (Pieris); Diacorpo; Single chain diabody; Single chain Fv fragments in tandem; TandAbs, Specific tries abs (Affimed); Darts (double affinity redirect; Macrogenics); Bispecific Xmabs (Xencor); Bispecific T cell couplers (Bites; Amgen; 55 kDa); Triplebodies; Derivatives of multifunctional recombinant antibodies of Fusion Protein (CreativeBiolabs) Tri-body (Fab-scFv); Duobody Platform (Genmab); Dock and lock platform; Knob into hole (KIH) platform; Bispecific humanized IgG antibody (REGN1979) (Regeneron); Bispecific Mab2 antibodies (F-Star); DVD-Ig (double variable domain immunoglobulin) (Abbvie); kappa-lambda bodies; TBTI (bispecific tetravalent tandem Ig); and CrossMab.

[0363] In certain embodiments, the bispecific polypeptide complex, as provided herein, is based on a bispecific format selected from bispecific IgG-type antibodies (BsIgG) comprising CrossMab; DAF (two in one); DAF (four in one); DutaMab; DT-IgG; Common LC knobs-into-holes; Knobs-into-holes assembly; Load pair; Fab arm change; SEEDbody; Triomab; Y-LIGHT; Fcab; kappa-lambda body; and orthogonal Fab. For a detailed description of bispecific antibody formats, see Spiess C., Zhai Q. and Carter PJ (2015) Molecular Immunology 67: 95-106, which is incorporated herein by reference in its entirety.

[0364] In certain embodiments, the bispecific polypeptide complex, as provided herein, is based on a bispecific format selected from antibodies attached to IgG with an additional antigen binding portion comprising DVD-IgG; IgG (H) -scFv; scFv- (H) IgG; IgG (L) -scFv; scFV- (L) IgG; IgG (L, H) -Fv; IgG (H) -V; V (H) -IgG; IgG (L) -V; V (L) -IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; and DVI-IgG (four in one) (see Id.).

[0365] In certain embodiments, the bispecific polypeptide complex, as provided herein, is based on a format selected from bispecific antibody fragments comprising Nanocorp; Nanocorp-HAS; BiTE; Diacorpo; DART; TandAb; scDiacorpo; sc-Diacorpo-CH3; Diacorpo-CH3; Triple Body; Mini-antibody; Minibody; TriBi Minibody; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F (ab ') 2; F (ab ') 2-scFv2; scFv-KIH; Fab-scFv-Fc; Tetravalent HCAb; scDiacorpo-Fc; Diacorpo-Fc; ScFv-Fc in tandem; and Intracorpo (see Id.).

[0366] In certain embodiments, the bispecific polypeptide complex as provided here is based on a bispecific format, such as Dock and Lock; ImmTAC; HSAcorpo; scDiacorpo-HAS; and scFv-Toxin in tandem (see Id.).

[0367] In certain embodiments, the bispecific polypeptide complex, as provided herein, is based on a format selected from bispecific antibody conjugates comprising IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2 (see Id.).

[0368] In certain embodiments, the first antigen binding portion and the second binding portion can be associated with an Ig-like structure. An Ig-like structure is like a natural antibody with a Y-shaped construction, with two arms for binding to the antigen and a trunk for association and stabilization. Similarity to the natural antibody can provide several advantages, such as good pharmacokinetics in vivo, desired immune response and stability, etc. It was found that the structure similar to

Ig comprising the first antigen binding portion provided here associated with the second antigen binding portion provided here exhibits thermal stability that is comparable to that of an Ig (e.g., an IgG). In certain embodiments, the Ig-like structure provided here is at least 70%, 80%, 90%, 95% or 100% that of a natural IgG.

[0369] In certain embodiments, the bispecific polypeptide complex comprises four polypeptide chains: i) VH1-C1-Hinge-CH2-CH3; ii) VL1- C2; iii) VH2-CH1-Hinge-CH2-CH3, and iv) VL2-CL, wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond, and the two hinge regions and / or the two CH3 domains are capable of forming one or more interchain links that can facilitate dimerization.

Antigenic specificities of the bispecific complex

[0370] The bispecific complex provided here has two antigenic specificities. The first and second antigen-binding portions are directed to the first and second antigen specificities, respectively.

[0371] The first and second antigenic specificities can be identical, in other words, the first and second antigen-binding portions bind to the same antigen molecule or the same epitope on the same antigen molecule.

[0372] Alternatively, the first and second antigenic specificities can be distinct. For example, the first and second antigen-binding portions can bind to different antigens. Such a bispecific polypeptide complex could be useful in, for example, bringing the two different antigens close together and thus promoting their interactions (for example, bringing immune cells very close to a tumor antigen or a pathogenic antigen and, therefore, promoting recognition or elimination of such an antigen by the immune system). For another example, the first and second antigen-binding portions can bind to different (and optionally non-overlapping) epitopes of an antigen. This can be useful for improving recognition or binding to a target antigen, in particular one that is susceptible to mutation (for example, a viral antigen).

[0373] In some embodiments, one of the bispecific complex antigen specificities provided here is directed at a specific T cell receptor molecule and / or a specific natural killer cell receptor molecule (NK cell). In some embodiments, one of the first and second antigen-binding moieties is capable of specifically binding to CD3, TCR, CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5 or CD95 and the other is capable of specifically bind to a tumor-associated antigen.

[0374] In certain embodiments, one of the antigenic specificities of the bispecific complex provided here is directed to CD3. In certain embodiments, the first antigen-binding portion of the bispecific complex is capable of specifically binding to CD3. In certain embodiments, the second antigen-binding portion of the bispecific complex is able to specifically bind to CD3.

[0375] In certain embodiments, the antigen-binding portion of the bispecific complexes comprises a VH1 and VL1 derived from an anti-CD3 antibody. In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein, wherein the first polypeptide and the second polypeptide comprise a pair of sequences selected from the group consisting of SEQ ID NOs: 2/1, 3/4 /, 5/1, 6/3, 7/3, 9/8, 10/8, 9/11, 10/11, 13/12, 15/14, 17/16, 17/18, 20/19, 21 / 12, 28/3, 3/29, 12/30, 12/31, 65/64, 67/66, 69/68, 70/68, 70/71, 72/71, 73/71, 75/74, 75/76, 78/77, 86/85, 90/89, 91/92 /, 94/93, 96/95, 98/97, 99/95, 101/100, 101/102, 106/105, 108 / 107, 110/109, 112/111, 137/136, 138/136,

137/139 and 138/139, where the variable regions of the anti-CD3 antibody (T3) are fused to the constant region of the TCR, as shown in Table 20.

[0376] In some embodiments, one of the bispecific complex antigen specificities provided here is directed at a specific T cell receptor molecule and / or a natural killer cell specific molecule (NK cell) and the other antigen specificity is addressed to a tumor-associated surface antigen. In certain embodiments, the first antigen-binding portion of the bispecific complex is capable of binding specifically to the specific T cell receptor molecule and / or to a specific natural killer cell (NK cell) receptor molecule (such as CD3) and to the second antigen the binding portion is able to specifically bind to a tumor-associated antigen (such as CD19) or vice versa.

[0377] In certain embodiments, bispecific polypeptide complexes comprise a combination of four sequences selected from the group consisting of SEQ ID NOs: 22/12/24/23 (E17, IgG1), 12/25/26/23 ( E17, IgG4) and 12/25/27/23 (F16), as shown in Example 8 and Table 20, where the first antigen binding portion binds CD3 and the second antigen binding portion binds CD19 . The E17 design is a bispecific and bivalent antibody, and the F16 design is a bispecific and trivalent antigen binding complex, with two Fab replications of the anti-CD19 antibody.

[0378] In certain embodiments, the bispecific polypeptide complex comprises a first antigen-binding portion that binds to CTLA-4, and a second antigen-binding portion that binds to PD-1, or vice versa.

[0379] In certain embodiments, the bispecific polypeptide complex comprises four polypeptide chains comprising: i) VH1 operably linked to a first chimeric constant region; ii) VL1 operationally linked to a second chimeric constant region; iii) VH2 operationally linked to the constant region of the conventional antibody heavy chain and iv) VL2 operationally linked to the constant region of the conventional antibody light chain. In certain embodiments, the first chimeric constant region can comprise C1- hinge-CH2-CH3, as defined above. In certain embodiments, the second chimeric constant region can comprise C2, as defined above.

In certain embodiments, the conventional antibody heavy chain constant region may comprise CH1-hinge-CH2-CH3, as defined above.

In certain embodiments, the light chain constant region of the conventional antibody may comprise CL, as defined above.

[0380] The following construction names are used interchangeably in this disclosure: E17-Design_2-QQQQ and W3438-T3U4.E17-1.uIgG4.SP; F16-Design-2- QQQQ and W3438-T3U4.F16-1.uIgG4.SP; U6T5.G25.IgG4 and W3248-U6T5.G25-

1.uIgG4.SP; and U6T1.G25R.IgG4 and W3248-U6T1.G25R-1.uIgG4.SP.

Preparation method

[0381] The present disclosure provides isolated nucleic acids or polynucleotides that encode the polypeptide complex, and the bispecific polypeptide complex provided herein.

[0382] The term "nucleic acid" or "polynucleotide", as used in this document, refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and their polymers in the form of single or double strands. Unless specifically limited, the term encompasses polynucleotides containing known natural nucleotide analogs that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific polynucleotide sequence also implicitly includes conservatively modified variants (for example, degenerate codon substitutions), alleles, orthologists, SNPs and complementary sequences, as well as the sequence explicitly indicated. Specifically, substitutions of degenerate codons can be achieved by generating sequences, in which the third position of one or more (or all) selected codons is replaced by mixed base residues and / or deoxinosine (see Batzer et al., Nucleic Acid Res 19: 5081 (1991); Ohtsuka et al., J.

Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)).

[0383] The nucleic acids or polynucleotides that encode the polypeptide complex and the bispecific polypeptide complex provided here can be constructed using recombinant techniques. To that end, the DNA encoding an antigen-binding portion of a parental antibody (such as CDR or variable region) can be isolated and sequenced using conventional procedures (for example, using oligonucleotide probes capable of specifically binding genes encoding the heavy and light chains of the antibody). Likewise, DNA encoding a TCR constant region can also be obtained. As an example, the polynucleotide sequence that encodes the variable domain (VH) and the polynucleotide sequence that encodes the first constant region (C1) of the TCR are obtained and operationally linked to allow transcription and expression in a host cell for production of the first polypeptide. Likewise, the VL encoding polynucleotide sequence is operably linked to the C1 encoding polynucleotide sequence, so as to allow expression of the second polypeptide in the host cell. If necessary, the polynucleotide sequences that encode one or more spacers are also operationally linked to the other coding sequences in order to allow expression of the desired product.

[0384] The coding polynucleotide sequences can further be operationally linked to one or more regulatory sequences, optionally in an expression vector, such that the expression or production of the first and second polypeptides is viable and under adequate control.

[0385] The polynucleotide sequence (s) encoding (s) can be inserted in a vector for later cloning (amplification of the DNA) or for expression, using recombinant techniques known in the state of art. In another embodiment, the polypeptide complex and the bispecific polypeptide complex provided herein can be produced by homologous recombination known in the art. Many vectors are available. The components of the vector generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (for example, SV40, CMV, EF-1α ) and a transcription completion sequence.

[0386] The term "vector", as used in this document, refers to a vehicle in which a polynucleotide encoding a protein can be operationally inserted in order to cause the expression of that protein.

Typically, the construct also includes appropriate regulatory sequences. For example, the polynucleotide molecule may include regulatory sequences located in the 5 'flanking region of the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding a site-directed modifier polypeptide, operably linked to the coding sequences in a manner capable of expressing the desired transcript / gene in a host cell. A vector can be used to transform, transduce or transfect a host cell, in order to cause the expression of the genetic element it carries within the host cell.

Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes, such as the yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage and virus from animals. The categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses and papovaviruses (eg SV40). A vector can contain a variety of elements to control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements and reporter genes. In addition, the vector may contain a source of replication. A vector may also include materials to assist its entry into the cell, including, but not limited to, a viral particle, a liposome or a protein coating.

[0387] In some embodiments, the vector system includes yeast, mammalian, bacterial systems, etc. and comprises plasmids such as, but not limited to, PALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDU, pDO , Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2 etc., and other laboratory and commercially available vectors. Suitable vectors can include, plasmid, or viral vectors (for example, retrovirus, adenovirus and adeno-associated defective replication viruses).

[0388] Vectors comprising the polynucleotide sequence (s) provided herein can be introduced into a host cell for cloning or gene expression. The phrase "host cell", as used herein, refers to a cell in which an exogenous polynucleotide and / or a vector has been introduced.

[0389] The host cells suitable for cloning or expressing DNA in the vectors cited here are the prokaryotic, yeast or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia,

Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans and Shigella, as well as bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0390] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for vectors encoding the polypeptide complex and the bispecific polypeptide complex. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, several other genera, species and strains are commonly available and useful here, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as, for example, K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K.

wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183 070); Candida; Trichoderma reesia (EP 244 234); Neurospora crassa; Schwanniomyces, like Schwanniomyces occidentalis; and filamentous fungi, such as hosts Neurospora, Penicillium, Tolypocladium and Aspergillus, such as A. nidulans and A. niger.

[0391] Host cells suitable for the expression of the glycosylated polypeptide complex, the bispecific polypeptide complex provided here is derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous strains and baculoviral variants and host cells of corresponding permissive insect hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and these viruses can be used as the virus according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Cultures of plant cells from cotton, corn, potatoes, soybeans, petunia, tomatoes and tobacco can also be used as hosts.

[0392] However, interest has been greater in vertebrate cells and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lineage (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), as Expi293; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol Reprod 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a strain of human hepatoma (Hep G2).

[0393] Host cells are transformed with the expression described above or the cloning vectors can be grown in conventional modified nutrient medium as appropriate to induce promoters, select transformants or amplify the cloning vectors.

[0394] For production of the polypeptide complex and the bispecific polypeptide complex provided here, host cells transformed with the expression vector can be cultured in a variety of media. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium (MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco Modified Eagle Medium (DMEM), Sigma) are suitable for cultivating host cells. In addition, any of the means described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem.

102: 255 (1980), US Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (like insulin, transferrin or epidermal growth factor), salts (like sodium chloride, calcium, magnesium and phosphate), buffers (like HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCINTM), trace elements (defined as inorganic compounds generally present in final concentrations in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements can also be included in appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH and the like, are those used previously with the host cell selected for expression and will be evident to the person skilled in the art.

[0395] In one aspect, the present disclosure provides a method of expressing the polypeptide complex and the bispecific polypeptide complex provided herein, which comprises culturing the host cell provided here under the condition that the polypeptide complex, or the polypeptide complex bispecific is expressed.

[0396] In certain embodiments, the present disclosure provides a method of producing the polypeptide complex provided herein, which comprises a) introducing into a host cell: a first polynucleotide encoding a first polypeptide comprising, from the N-terminus to the Ç-

terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the TCR and a second polynucleotide encoding a second polypeptide comprising, from the N-terminal to the C-terminal end, a first light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, where: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between C1 and C2, and the non-native interchain bond is able to stabilize the dimer, and the first antibody has a first antigen specificity; b) allowing the host cell to express the polypeptide complex.

[0397] In certain embodiments, the present disclosure provides a method of producing the bispecific polypeptide complex provided herein, which comprises a) introducing into a host cell: a first polynucleotide encoding a first polypeptide comprising, from the N-terminus to the C-terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the TCR, a second polynucleotide encoding a second polypeptide comprising, from the N-terminal end to the C- terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, and one or more additional polynucleotides encoding a second antigen binding portion, where: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain bond between a first mutated residue comprised in C1 and an s According to the mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer of C1 and C2, the first antigen binding portion and the second antigen binding portion reduced the mismatch of what would otherwise be the case. the first antigen-binding portion was equivalent to the natural Fab, and the first antibody had a first antigenic specificity and the second antibody had a second antigenic specificity, b) allowing the host cell to express the bispecific polypeptide complex.

[0398] In certain embodiments, the method further comprises isolating the polypeptide complex.

[0399] When using recombinant techniques, the polypeptide complex, the bispecific polypeptide complex provided here can be produced intracellularly, in the periplasmic space, or secreted directly into the medium. If the product is produced intracellularly, as a first step, particulate debris, host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe a procedure to isolate antibodies that are secreted into the E. coli periplasmic space. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 min. Cell debris can be removed by centrifugation. When the product is secreted in the medium, the supernatants of such expression systems are, in general, first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, can be included in any of the previous steps to inhibit proteolysis and antibiotics can be included to prevent the growth of foreign contaminants.

[0400] The polypeptide complex and bispecific polypeptide complex provided here from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulphate precipitation, salting out and affinity chromatography, affinity chromatography being the preferred purification technique.

[0401] Where the polypeptide complex or the bispecific polypeptide complex provided here comprises the immunoglobulin Fc domain, then protein A can be used as an affinity linker, depending on the species and isotype of the Fc domain that is present in the polypeptide complex . Protein A can be used for the purification of polypeptide complexes based on human γ1, γ2, or y4 heavy chains (Lindmark et al., J. Immunol Meth 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 1567 1575 (1986)). The matrix to which the affinity linker is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenodivinyl) benzene allow for faster flow rates and shorter processing times than those obtained with agarose.

[0402] Where the polypeptide complex or the bispecific polypeptide complex provided here comprises a CH3 domain, the Bakerbond ABX resin (J.T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, SEPHAROSETM heparin chromatography, anionic or cationic exchange chromatography (such as a polyaspartic acid column) , chromato-focusing, SDS-PAGE and precipitation with ammonium sulfate are also available depending on the antibody to be recovered.

[0403] After which preliminary purification step (s), the mixture comprising the polypeptide complex of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably carried out at low concentrations of salt (for example, about 0-0.25 M of salt).

[0404] In certain embodiments, the bispecific polypeptide complex provided here can be easily purified in high yields using conventional methods. One of the advantages of the bispecific polypeptide complex is the significantly reduced mismatch between the heavy and light chain variable domain sequences. This reduces the production of unwanted by-products and makes it possible to obtain high-purity products with high yields through the use of relatively simple purification processes.

Derivatives

[0405] In certain embodiments, the polypeptide complex or the bispecific polypeptide complex can be used as the base of conjugation with the desired conjugates.

[0406] It is understood that a variety of conjugates may be linked to the polypeptide complex or the bispecific polypeptide complex provided here (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. ( eds.), Carger Press, New York, (1989)).

These conjugates can be linked to the polypeptide complex or the bispecific polypeptide complex by covalent bonding, affinity bonding, intercalation, coordinated bonding, complexation, association, mixing or addition, among other methods.

[0407] In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein can be modified to contain specific sites outside the epitope-binding portion that can be used for binding to one or more conjugates. For example, that site may include one or more reactive amino acid residues, such as, for example, cysteine or histidine residues, to facilitate covalent binding to a conjugate.

[0408] In certain embodiments, the polypeptide complex or the bispecific polypeptide complex can be linked to a conjugate indirectly, or indirectly, for example, through another conjugate or through a linker.

[0409] For example, the polypeptide complex or the bispecific polypeptide complex with a reactive residue, such as cysteine, can be attached to a thiol-reactive agent in which the reactive group is, for example, a maleimide, an iodoacetamide, a pyridyl disulfide, or other thiol-reactive conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc .; Brinkley, 1992, Bioconjugate Chem. 3: 2; Garman, 1997, Non-Radioactive Labeling : A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1: 2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0410] For another example, the polypeptide complex or the bispecific polypeptide complex can be conjugated to biotin, then indirectly conjugated to a second conjugate which is conjugated to avidin. In yet another example, the polypeptide complex or the bispecific polypeptide complex can be linked to a linker that still binds to the conjugate. Examples of binders include bifunctional coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT) , bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as dissuccinimidyl suherate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and hisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-

737 (1978)) and N-succinimidyl-4- (2-pyridylthio)) pentanoate (SPP) to provide a disulfide bond.

[0411] The conjugate can be a detectable marker, a pharmacokinetic modification portion, a purification portion or a cytotoxic portion.

Examples of detectable markers may include fluorescent markers (eg, fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme-substrate markers (eg, horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase), radioisotopes (e.g. 123I, 124I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm , 212Bi, and 32P, other lanthanides, luminescent markers), chromophoric portion, digoxigenin, biotin / avidin, DNA molecule or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying portion, such as PEG, which helps to increase the half-life of the antibody. Other suitable polymers include, such as carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers and the like. In certain embodiments, the conjugate may be a purification portion, such as a magnetic sphere. A "cytotoxic portion" can be any agent that is harmful to cells or that can damage or kill cells. Examples of the cytotoxic portion include, without limitation, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracine dione, mitoxantrone, mitram, mitram actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine agents) example, mecloretamine, thiope chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,

streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin and amthramycin )) and antithymotic agents (for example, vincristine and vinblastine).

[0412] Methods for conjugating conjugates to proteins such as antibodies, immunoglobulins or fragments thereof are found, for example, in U.S. Patent No. 5,208,020; Pat. No. 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488, which are incorporated herein by reference in their entirety.

Pharmaceutical composition

[0413] The present disclosure also provides a pharmaceutical composition comprising the polypeptide complex or the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.

[0414] The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) and / or salt is, in general, chemically and / or physically compatible with the other ingredients that comprise the formulation and is physiologically compatible with the receiver of it.

[0415] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is not an active ingredient, that has acceptable bioactivity and is not toxic to an individual. Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, agents sequestering or chelating agents, diluents, adjuvants, excipients or pharmaceutically acceptable non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0416] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, dyes, emulsifiers or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene and / or propyl gallate. As disclosed in this document, the inclusion of one or more antioxidants such as methionine in a pharmaceutical composition provided herein decreases the oxidation of the polypeptide complex or the bispecific polypeptide complex. This reduction in oxidation prevents or reduces the loss of binding affinity, improving protein stability and maximizing shelf life. Therefore, in certain embodiments, compositions are provided that comprise the polypeptide complex or the bispecific polypeptide complex disclosed herein and one or more antioxidants, such as methionine.

[0417] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles, such as sodium chloride injection, Ringer injection, isotonic dextrose injection, sterile water injection or Ringer injection with dextrose and lactate, vehicles non-aqueous, such as fixed oils of vegetable origin, cotton oil, corn oil, sesame oil or peanut oil, antimicrobial agents in bacteriostatic or fungistatic concentrations, isotonic agents, such as sodium chloride or dextrose, buffers, such as phosphate buffers or citrate, antioxidants, such as sodium bisulfate, local anesthetics, such as procaine hydrochloride, suspending and dispersing agents, such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents, such as Polysorbate 80 (TWEEN-80), sequestering agents or chelating agents such as

EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylenolicol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multiple dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl esters of p-hydroxybenzoic acid, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0418] The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, prolonged release formulation or powder. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0419] In certain embodiments, the pharmaceutical compositions are formulated into an injectable composition. Injectable pharmaceutical compositions can be prepared in any conventional form, such as, for example, liquid solution, suspension, emulsion or solid forms suitable for generating the liquid solution, suspension or emulsion. Injection preparations may include sterile and / or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent immediately before use, including hypodermic tablets, sterile suspensions ready for injection, sterile products insoluble dry matter ready to be combined with a vehicle immediately before use and sterile and / or non-pyretic emulsions. The solutions can be aqueous or non-aqueous.

[0420] In certain embodiments, parenteral preparations in a single dose are packaged in an ampoule, vial or syringe with a needle.

All preparations for parenteral administration must be sterile and antipyretic, as is known and practiced in the state of the art.

[0421] In certain embodiments, a sterile and lyophilized powder is prepared by dissolving the polypeptide complex or the bispecific polypeptide complex, as disclosed here, in a suitable solvent. The solvent may contain an excipient that improves the stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other buffer known to those skilled in the art, in an embodiment, at approximately neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art generates a desirable formulation. In one embodiment, the resulting solution will be distributed in vials for lyophilization. Each vial may contain a single dosage or multiple dosages of the polypeptide complex, the bispecific polypeptide complex provided here or its composition. Overfilling vials with a small amount above that needed for one dose or set of doses (for example, about 10%) is acceptable, in order to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as about 4 ° C to room temperature.

[0422] Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution, sterile and / or non-pyretic water or another suitable liquid carrier is added to the lyophilized powder. The precise amount depends on the therapy selected and can be determined empirically.

Treatment method

[0423] Therapeutic methods are also provided, which include: administering an effective amount of the polypeptide complex or the bispecific polypeptide complex provided here to an individual who needs it, treating or preventing a condition or disorder. In certain embodiments, the individual has been identified as having a disorder or condition liable to respond to the polypeptide complex or the bispecific polypeptide complex provided here.

[0424] "Treating" or "treating" a condition, as used here, includes preventing or alleviating a condition, delaying the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the developing symptoms associated with a condition, reducing or eliminating symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition or some combination thereof.

[0425] The therapeutically effective amount of the polypeptide complex and the bispecific polypeptide complex provided here will depend on several factors known in the state of the art, such as, for example, body weight, age, past medical history, current medications, patient's health status and potential for cross-reaction, allergies, sensitivities and adverse side effects, as well as the route of administration and extent of disease development. Dosages can be proportionally reduced or increased by a person skilled in the art (for example, doctor or veterinarian), as indicated by these and other circumstances or requirements.

[0426] In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein can be administered in a therapeutically effective dosage of about 0.01 mg / kg to about 100 mg / kg (for example, about 0 , 01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg or about 100 mg / kg). In some of these embodiments, the polypeptide complex or the bispecific polypeptide complex provided here is administered at a dosage of about 50 mg / kg or less, and in some of these embodiments the dosage is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dosage of administration may change over the course of treatment. For example, in certain embodiments, the initial administration dosage may be greater than the subsequent administration dosages. In certain embodiments, the dosage of administration may vary over the course of treatment, depending on the individual's reaction.

[0427] Dosage regimens can be adjusted to provide the ideal desired response (for example, a therapeutic response). For example, a single dose can be administered or several divided doses can be administered over time.

[0428] The polypeptide complex or the bispecific polypeptide complex provided herein can be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal,

intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (for example, oral, intranasal, intraocular, sublingual, rectal or topical).

[0429] In certain embodiments, the condition or disorder treated by the polypeptide complex or the bispecific polypeptide complex provided here is cancer or cancerous condition, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neuropathies, neuropsychiatric conditions, injuries, inflammations , or clotting disorder.

[0430] "Cancer" or "cancerous condition", as used in this document, refers to any medical condition mediated by the growth, proliferation or metastasis of neoplastic or malignant cells, and includes solid cancers and non-solid cancers, such as leukemia. “Tumor”, as used in this document, refers to a solid mass of neoplastic and / or malignant cells.

[0431] With respect to cancer, "treating" or "treatment" may refer to the inhibition or slowing of the growth, proliferation or metastasis of neoplastic or malignant cells, preventing or delaying the development of growth, proliferation or metastasis of neoplastic or malignant cells , or some combination thereof. With respect to a tumor, “treating” or “treating” includes eradicating all or part of a tumor, inhibiting or slowing the growth and metastasis of the tumor, preventing or delaying the development of a tumor, or some combination of themselves.

[0432] For example, with respect to the use of the polypeptide complex or bispecific polypeptide complex disclosed here to treat cancer, a therapeutically effective amount is the dosage or concentration of the polypeptide complex capable of eradicating all or part of a tumor, inhibiting or slowing down the tumor growth, inhibiting cell growth or proliferation mediating a cancerous condition, inhibiting tumor cell metastasis, improving any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition or any combination thereof.

[0433] In certain embodiments, conditions and disorders include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, cancer of breast, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, fungal mycoses, Merkel cell cancer and other malignant hematological diseases, such as classic Hodgkin's lymphoma (CHL), primary large mediastinal cell lymphoma, B cell lymphoma / histiocyte / T cell rich, positive PTLD and negative for EBV and diffuse large B-cell lymphoma associated with EBV (DLBCL), plasmablastic lymphoma, extranodal NK / T cell lymphoma, nasopharyngeal carcinoma and primary stroke lymphoma associated with HHV8, lymphoma Hodgkin's disease, neoplasm of the central nervous system (CNS), as primary CNS lymphoma, tumor in the spinal axis, brain stem glioma.

[0434] In certain embodiments, conditions and disorders include a CD19-related disease or condition, such as B-cell lymphoma, optionally Hodgkin's lymphoma or non-Hodgkin's lymphoma, in which non-Hodgkin's lymphoma comprises: Diffuse large cell lymphoma B (DLBCL), follicular lymphoma, marginal zone B cell lymphoma (MZL), mucosa-associated lymphatic tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, LLC) or mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL) or Waldenstrom's macroglobulinemia (WM).

[0435] In certain embodiments, conditions and disorders include hyperproliferative conditions or infectious diseases that can be treated by regulating the immune response by CTLA-4 and / or PD-1. Examples of hyperproliferative conditions include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors and metastatic lesions.

[0436] The polypeptide complex or bispecific polypeptide complex can be administered alone or in combination with one or more additional therapeutic means or agents.

[0437] In certain embodiments, when used to treat cancer or tumor or proliferative disease, the polypeptide complex or the bispecific polypeptide complex provided here can be administered in combination with chemotherapy, radiation therapy, surgery for the treatment of cancer (for example, tumorectomy), one or more antiemetics or other treatments for complications from chemotherapy or any other therapeutic agent for use in the treatment of cancer or any related medical disorder. “Administered in combination”, as used in this document, includes administering simultaneously as part of the same pharmaceutical composition, at the same time as separate compositions, or at different times as separate compositions. A composition that is administered before or after another agent is considered to be administered "in combination" with that agent, according to the phrase used in this document, even if the composition and the second agent are administered by different routes. When possible, additional therapeutic agents administered in combination with the polypeptide complex or the bispecific polypeptide complex provided here are administered according to the schedule listed in the product information sheet for the additional therapeutic agent, or according to the Physicians' Desk Reference, 70th Edition (2016) or protocols well known in the state of the art.

[0438] In certain embodiments, therapeutic agents can induce or increase the immune response against cancer. For example, a tumor vaccine can be used to induce an immune response to certain tumors or cancer. Cytokine therapy can also be used to improve the presentation of tumor antigens to the immune system. Examples of cytokine therapy include, without limitation, interferons, such as interferon-α, -β and -γ, colony-stimulating factors, such as macrophage CSF, macrophage and granulocyte CSF, granulocyte CSF, interleukins such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, factors of tumor necrosis, such as TNF-α and TNF-β. Agents that inactivate immunosuppressive targets can also be used, for example, TGF-beta inhibitors, IL-10 inhibitors and Fas ligand inhibitors.

Another group of agents includes those that activate the immune response against tumor or cancer cells, for example, by increasing the activation of T cells (for example, agonist of T cell co-stimulatory molecules, such as CTLA-4, ICOS and OX-40), and increasing the function of the dendritic cell and the presentation of antigens.

Kits

[0439] The present disclosure further provides kits comprising the polypeptide complex or the bispecific polypeptide complex provided here.

In some embodiments, the kits are useful for detecting the presence or level of, or capturing or enriching, one or more targets of interest in a biological sample. The biological sample can comprise a cell or tissue.

[0440] In some examples, the kit comprises the polypeptide complex or the bispecific polypeptide complex provided here, which is conjugated to a detectable marker. In certain other embodiments, the kit comprises a non-labeled polypeptide complex or the non-labeled bispecific polypeptide complex provided herein and further comprises a secondary labeled antibody that is capable of binding to the non-labeled polypeptide complex or bispecific polypeptide complex provided here. The kit may also comprise an instruction for use and a packaging that separates each of the components in the kit.

[0441] In certain embodiments, the polypeptide complex or the bispecific polypeptide complex provided herein is associated with a substrate or device. Substrate or useful device can be, for example, magnetic beads, microtiter plates, or test strip. These can be useful for a binding assay (such as ELISA), an immunographic assay, capture or enrichment of a target molecule in a biological sample.

[0442] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention.

All specific compositions, materials and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials and methods are not intended to limit the invention, but only to illustrate specific embodiments that fall within the scope of the invention. A person skilled in the art can develop equivalent compositions, materials and methods without exercising inventive capacity and without departing from the scope of the invention. It should be understood that many variations can be made in the procedures described here while still remaining within the limits of the present invention. It is the inventors' intention that such variations are included within the scope of the invention.

TABLE 11. Drawings and names of the chimeric constant regions (CBeta / CAlfa) SEQ ID NOs: Model drawings based on the Table (Heavy Chain (HC) / Light Chain (LC)) - 16 IgG1 Design_1 177/176 Design_2 179/178 Design_3 184 / 183 Design_4 185/183 Design_5 180/176

SEQ ID NOs: Model drawings based on the Table (Heavy Chain (HC) / Light Chain (LC)) - 16 IgG1

Design_6 181/178

Design_6a 182/178

Design_7 184/186

Design_8 185/186

TABLE 12. Domains and SEQ ID NOs. of the chimeric constant regions

(CBeta / CAlfa) Region name Domains from the N-terminus to the C-terminus and their SEQ ID NOs. constant First or Third domain of chimera and SEQ Constant domain of the domain of second domain junction + hinge ID NOs. of TCR (C1 or C2) Dimerization (D) of junction (CJ) (CJ ') chain Design_1 HC HCJ1 CBeta (S56C) CJ'1G1 FcG1 SEQ ID NO: 177 SEQ ID NO: 49 SEQ ID NO: 33 SEQ ID NO : 53 SEQ ID NO: 302 Design_1 LC LCJ1 CAlpha (T49C) SEQ ID NO: 176 SEQ ID NO: 51 SEQ ID NO: 43 Design_2 HC HCJ2 CBeta (S56C) CJ'1G1 FcG1 SEQ ID NO: 179 SEQ ID NO: 50 SEQ ID NO: 33 SEQ ID NO: 53 SEQ ID NO: 302 Design_2 LC LCJ2 CAlpha (T49C) SEQ ID NO: 178 SEQ ID NO: 52 SEQ ID NO: 43 Design_3 HC HCJ3 CAlpha (T49C) CJ'2G1 FcG1 SEQ ID NO: 184 SEQ ID NO: 129 SEQ ID NO: 43 SEQ ID NO: 134 SEQ ID NO: 302 Design_3 LC LCJ3 CBeta (S56C) SEQ ID NO: 33 + SEQ ID NO: 183 SEQ ID NO: 308 NO: 306 Design_4 HC HCJ4 CAlpha (T49C) CJ'2G1 FcG1 SEQ ID NO: 185 SEQ ID NO: 130 SEQ ID NO: 43 SEQ ID NO: 302 SEQ ID NO: 302 Design_4 LC LCJ3 CBeta (S56C) SEQ ID NO: 33 + SEQ ID NO: 183 SEQ ID NO: 308 NO: 306 Design_5 HC HCJ1 CBeta (S56C) (FG-) CJ'1G1 FcG1 SEQ ID NO: 180 SEQ ID NO: 49 SEQ ID NO: 37 SEQ ID NO: 53 SEQ ID NO: 302 Design_5 LC LCJ1 CAlpha (T49C) SEQ ID NO: 176 SEQ ID NO: 51 SEQ ID NO: 4 3 Design_6 HC HCJ1 CBeta (S56C) (FG-) CJ'1G1 FcG1 SEQ ID NO: 181 SEQ ID NO: 50 SEQ ID NO: 53 SEQ ID NO: 53 SEQ ID NO: 302 Design_6 LC LCJ2 CAlpha (T49C) SEQ ID NO: 178 SEQ ID NO: 52 SEQ ID NO: 43 Design_6a HC HCJ2 CBeta (S56C) (DE-FG-) CJ'1G1 FcG1 SEQ ID NO: 182 SEQ ID NO: 50 SEQ ID NO: 41 SEQ ID NO: 53 SEQ ID NO: 302 Design_6a LC LCJ2 CAlpha (T49C) SEQ ID NO: 178 SEQ ID NO: 52 SEQ ID NO: 43 Design_7 HC HCJ3 CAlpha (T49C) CJ'2G1 FcG1 SEQ ID NO: 184 SEQ ID NO: 129 SEQ ID NO: 43 SEQ ID NO: 134 SEQ ID NO: 302 Design_7 LC LCJ3 CBeta (S56C) (FG-) SEQ ID NO: 37 + SEQ ID NO: 186 SEQ ID NO: 308 NO: 306 Design_8 HC HCJ4 CAlpha (T49C) CJ'2G1 FcG1 SEQ ID NO: 185 SEQ ID NO: 130 SEQ ID NO: 43 SEQ ID NO: 134 SEQ ID NO: 302

Region name Domains from the N-terminus to the C-terminus and their SEQ ID NOs. constant First or Third domain of chimera and SEQ Constant domain of the domain of second domain junction + hinge ID NOs. of TCR (C1 or C2) Dimerization (D) of junction (CJ) (CJ ') chain Design_8 LC LCJ3 CBeta (S56C) (FG-) SEQ ID NO: 37 + SEQ ID NO: 186 SEQ ID NO: 308 NO: 306

TABLE 13. Designs and names of Design_2 (CBeta / CAlfa) without glycosylation sites SEQ ID NO: (HC-CBeta / LC-CAlfa) Sample HC / LC 188/187 (IgG1) Design_2-QQQQ 196/187 (IgG4)

Design_2-AAAA 190/189 (IgG1)

Design_2-QSKE 192/191 (IgG1)

Design_2-ASKE 192/193 (IgG1)

Design_2-QQQQQ 195/194 (IgG1)

TABLE 14. Domains and SEQ ID NOs. do Design_2 (CBeta / CAlfa) without glycosylation sites Domain Name from the N-terminal to the C-terminal and their SEQ ID NOs. complex and First or Third domain SEQ ID Domain constant of the domain of second junction domain + NOs. of TCR (C1 or C2) Dimensioning (D) hinge joint (CJ ') chain: Design_2- QQQQ HCJ2 CBeta (S56C) (N69Q) CJ'1G1 FcG1 (IgG1) HC SEQ ID NO: SEQ ID NO: 50 SEQ ID NO: 34 SEQ ID NO: 53 SEQ ID NO: 302 188 Design_2- CAlpha (T49C) QQQQ LCJ2 (N34Q + N68Q + N79Q) (IgG1) LC SEQ ID NO: SEQ ID NO: 52 SEQ ID NO: 44 187 Design_2- QQQQ HCJ2 CBeta (S56C) (N69Q) CJ'1G4 FcG4 (IgG4) HC SEQ ID NO: SEQ ID NO: 50 SEQ ID NO: 34 SEQ ID NO: 54 SEQ ID NO: 303 196 Design_2- CAlpha (T49C) QQQQ LCJ2 (N34Q + N68Q + N79Q) (IgG4) LC SEQ ID NO: SEQ ID NO: 52 SEQ ID NO: 44 187

Domain Name from the N-terminal to the C-terminal end and its SEQ ID NOs. complex and First or Third domain SEQ ID Domain constant of the domain of second junction domain + NOs. of TCR (C1 or C2) Dimensioning (D) hinge joint (CJ ') chain: Design_2- AAAA HCJ2 CBeta (S56C) (N69A) CJ'1G1 FcG1 (IgG1) HC SEQ ID NO: SEQ ID NO: 50 SEQ ID NO: 35 SEQ ID NO: 53 SEQ ID NO: 302 190 Design_2- CAlpha (T49C) AAAA LCJ2 (N34A + N68A + N79A) (IgG1) LC SEQ ID NO: SEQ ID NO: 52 SEQ ID NO: 45 189 Design_2- QSKE HCJ2 CBeta (S56C) (N69E) CJ'1G1 FcG1 (IgG1) HC SEQ ID NO: SEQ ID NO: 50 SEQ ID NO: 36 SEQ ID NO: 53 SEQ ID NO: 302 192 Design_2- CAlpha (T49C) QSKE LCJ2 (N34Q + N68S + N79K) (IgG1) LC SEQ ID NO: SEQ ID NO: 52 SEQ ID NO: 46 191 Design_2- ASKE HCJ2 CBeta (S56C) (N69E) CJ'1G1 FcG1 (IgG1) HC SEQ ID NO: SEQ ID NO: 50 SEQ ID NO: 36 SEQ ID NO: 53 SEQ ID NO: 302 192 Design_2- CAlpha (T49C) ASKE LCJ2 (N34A + N68S + N79K) (IgG1) LC SEQ ID NO: SEQ ID NO: 52 SEQ ID NO: 47 191 Design_2- QQQQQ HCJ2 CBeta (S56C) (N69Q) CJ'1G1 FcG1 (IgG1) HC SEQ ID NO: SEQ ID NO: 50 SEQ ID NO: 34 SEQ ID NO: 53 SEQ ID NO: 302 195 Design_2- CAlpha (T49C) QQQQQ LCJ2 (N34Q + N68Q + (IgG1) LC N79Q + N61Q) SEQ ID NO: SEQ ID NO: 52 SEQ I D NO: 48 194

TABLE 15. Drawings and names of the chimeric constant regions

(CBeta / CPré-Alpha) Based models Sequence file SEQ ID NOs: in Table 16 (IgG1) Design_1_Pre_TCR_ Conjunction'1 198/197 Design_2_Pre_TCR_Conjunction'_1_Cys10 200/199 Design_3_Pre_TCR_Conjunction'_1_Cys11 202/201 Design_4_Pre_TCR_Con Design_5_Pre_TCR_Conjunction'_1_Cys13 203/204 Design_6_Pre_TCR_Conjunction'_1_Cys14 205/204 Design_7_Pre_TCR_Conjunction'_1_Cys15 206/204 Design_8_Pre_TCR_Conjunction'_1_Cys1_4L4T_1 208/207

Design_9_Pre_TCR_Conjunction'_1_Cys2_4L4T_2 Design_10_Pre_TCR_Conjunction'_1_Cys4 208/209 211/210 213/212 TCR_Design_5_crossed_1 Pre-Pre-Pre-TCR_Design_C TCR_Design_6_crossed_1 TCR_Design_5_crossed_2 214/212 213/215 Pre-Pre-Pre-TCR_Design_D TCR_Design_6_crossed_2 214/215 Table 16. Domains and SEQ ID NOs . of the chimeric constant regions (CBeta / CPré-Alpha) Domain Name from the N-terminus to the C-terminus and their SEQ ID NOs. complex and SEQ First or Third domain Domain Domain contained in TCR ID NOs. of the second junction domain + Dimerization (C1 or C2) chain: hinge junction (CJ) (CJ ') (D) Design_1_Pre_T CR_Conjunction ’HCJB CBeta (N69Q) CJ'1G1 FcG1 1 HC

SEQ ID SEQ ID NO: 198 SEQ ID NO: 50 SEQ ID NO: 84 SEQ ID NO: 53 NO: 302 Design_1_Pre_T CR_ LCJB CPreAlpha (N50Q) Conjunction'1 LC SEQ ID NO: 197 SEQ ID NO: 309 SEQ ID NO: 83 Design_2_Pre_T CR_Conjunction 'HCJB CBeta (S76C) (N69Q) CJ'1G1 FcG1 _1_Cys10 HC

SEQ ID SEQ ID NO: 200 SEQ ID NO: 50 SEQ ID NO: 319 SEQ ID NO: 53 NO: 302 Design_2_Pre_T CR_Conjunction 'LCJB CPreAlpha (Y59C) (N50Q) _1_Cys10 LC SEQ ID NO: 199 SEQ ID NO: 309 SEQ ID NO: 309 NO: 311 Design_3_Pre_T CR_Conjunction 'HCJB CBeta (F13C) (N69Q) CJ'1G1 FcG1 _1_Cys11 HC

SEQ ID SEQ ID NO: 202 SEQ ID NO: 50 SEQ ID NO: 320 SEQ ID NO: 53 NO: 302 Design_3_Pre_T CR_Conjunction 'LCJB CPreAlpha (A13C) (N50Q) _1_Cys11 LC SEQ ID NO: 201 SEQ ID NO: 309 SEQ ID NO: 312 Design_4_Pre_T CR_Conjunction 'HCJB CBeta (S16C) (N69Q) CJ'1G1 FcG1 _1_Cys12 HC

SEQ ID SEQ ID NO: 203 SEQ ID NO: 50 SEQ ID NO: 321 SEQ ID NO: 53 NO: 302 Design_4_Pre_T CR_Conjunction 'LCJB CPreAlpha (A13C) (N50Q) _1_Cys12 LC SEQ ID NO: 201 SEQ ID NO: 309 SEQ ID NO: 312 Design_5_Pre_T CR_Conjunction 'HCJB CBeta (S16C) (N69Q) CJ'1G1 FcG1 _1_Cys13 HC

SEQ ID SEQ ID NO: 203 SEQ ID NO: 50 SEQ ID NO: 321 SEQ ID NO: 53 NO: 302 Design_5_Pre_T CR_Conjunction 'LCJB CPreAlpha (S11C) (N50Q) _1_Cys13 LC SEQ ID NO: 204 SEQ ID NO: 309 SEQ ID NO: 313

Domain Name from the N-terminal to the C-terminal end and its SEQ ID NOs. complex and SEQ First or Third domain Domain Domain contained in TCR ID NOs. of the second junction domain + Dimerization (C1 or C2) chain: hinge junction (CJ ') (D) Design_6_Pre_T CR_Conjunction ’HCJB CBeta (A18C) (N69Q) CJ'1G1 FcG1 _1_Cys14 HC

SEQ ID SEQ ID NO: 205 SEQ ID NO: 50 SEQ ID NO: 322 SEQ ID NO: 53 NO: 302 Design_6_Pre_T CR_Conjunction 'LCJB CPreAlpha (S11C) (N50Q) _1_Cys14 LC SEQ ID NO: 204 SEQ ID NO: 309 SEQ ID NO: 309 SEQ ID NO: 309 SEQ ID NO: 309 SEQ ID NO: 309 NO: 313 Design_7_Pre_T CR_Conjunction 'HCJB CBeta (E19C) (N69Q) CJ'1G1 FcG1 _1_Cys15 HC

SEQ ID SEQ ID NO: 206 SEQ ID NO: 50 SEQ ID NO: 323 SEQ ID NO: 53 NO: 302 Design_7_Pre_T CR_Conjunction 'LCJB CPreAlpha (S11C) (N50Q) _1_Cys15 LC SEQ ID NO: 204 SEQ ID NO: 309 SEQ ID NO: 309 NO: 313 Design_8_Pre_T CR_Conjunction 'HCJB CBeta (S56C) (N69Q) CJ'1G1 FcG1 _1_Cys1_4L4T_1

HC

SEQ ID SEQ ID NO: 208 SEQ ID NO: 50 SEQ ID NO: 34 SEQ ID NO: 53 NO: 302 Design_8_Pre_T CR_Conjunction ’LCJB CPreAlpha (S62C) (N50Q) _1_Cys1_4L4T_1

LC SEQ ID NO: 207 SEQ ID NO: 309 SEQ ID NO: 314 Design_9_Pre_T CR_Conjunction ’HCJB CBeta (S56C) (N69Q) CJ'1G1 FcG1 _1_Cys2_4L4T_2

HC

SEQ ID SEQ ID NO: 207 SEQ ID NO: 50 SEQ ID NO: 34 SEQ ID NO: 53 NO: 302 Design_9_Pre_T CR_Conjunction ’LCJB CPreAlpha (T65C) (N50Q) _1_Cys2_4L4T_2

LC SEQ ID NO: 209 SEQ ID NO: 309 SEQ ID NO: 315 Design_10_Pre_ TCR_Conjunction HCJB CBeta (A11C) (N69Q) CJ'1G1 FcG1 ’_1_Cys4 HC

SEQ ID SEQ ID NO: 211 SEQ ID NO: 50 SEQ ID NO: 324 SEQ ID NO: 53 NO: 302 Design_10_Pre_ TCR_Conjunction LCJB CPreAlpha (I16C) (N50Q) '_1_Cys4 LC SEQ ID NO: 210 SEQ ID NO: 309 SEQ ID NO: 309 NO: 316 Pre- TCR_Design_5_c rossed_1 HC SEQ ID NO: 213 HCJC CPreAlpha (S11C) (N50Q) CJ'2G1 FcG1 Pre-

SEQ ID TCR_Design_5_c SEQ ID NO: 132 SEQ ID NO: 313 SEQ ID NO: 134 NO: 302 rossed_1 LC SEQ ID NO: 212 LCJC Cbeta (N69Q, S16C)

Domain Name from the N-terminal to the C-terminal end and its SEQ ID NOs. complex and SEQ First or Third domain Domain Domain contained in TCR ID NOs. of the second junction domain + Dimerization (C1 or C2) chain: hinge junction (CJ ') (D) Pre- TCR_Design_6_c SEQ ID NO: 50 SEQ ID NO: 321+ 306 rossed_1 HC SEQ ID NO: 213 Pre - TCR_Design_6_c HCJC CPreAlpha (S11C) (N50Q) CJ'2G4 FcG4 rossed_1 LC

SEQ ID SEQ ID NO: 215 SEQ ID NO: 132 SEQ ID NO: 313 SEQ ID NO: 134 NO: 303 Pre- TCR_Design_5_c LCJC CBeta (N69Q, A18C) rossed_2 HC SEQ ID NO: 214 SEQ ID NO: 50 SEQ ID NO: 50 SEQ ID NO: 50 SEQ ID NO: 50 : 322+ 306 Pre- TCR_Design_5_c rossed_2 LC SEQ ID NO: 212 HCJD CPreAlpha (S11C) (N50Q) CJ'2G1 FcG1 Pre-

SEQ ID TCR_Design_6_c SEQ ID NO: 133 SEQ ID NO: 313 SEQ ID NO: 134 NO: 302 rossed_2 HC SEQ ID NO: 214 LCJC Cbeta (N69Q, S16C) Pre- TCR_Design_6_c SEQ ID NO: 50 SEQ ID NO: NO: 321 + 306 rossed_2 LC SEQ ID NO: 215 HCJD CPreAlpha (S11C) (N50Q) CJ'2G1 FcG1 TABLE 17. Drawings and names of the chimeric constant regions (CGama / CDelta) Models based Construction of SEQ ID NOs design in HC / LC in the Table 13 (IgG1) dg_Design_1 dg_Design_1 dg_Design_2 234/233 232/231 216/215 dg_Design_2_no_Glyco dg_Design_2_hypeCys1_no_Glyco dg_Design_2_hypeCys2_no_Glyco 218/217 220/219 222/221 dg_Design_2_hypeCys3_no_Glyco dg_Design_2 dg_Design_2_Cys2_no_Glyco dg_Design_2_Cys1_no_Glyco 224/223 226/225 227/223 dg_Design_2_Cys3_no_Glyco dg_Design_2_Cys4_no_Glyco 229/228 229/230 dg_Design_2_Cys5_no_Glyco dg_Design_3 dg_crossed_Design_1 236/235 dg_Design_4 dg_crossed_Deisng_2 238/237 TABLE 18. Domains and SEQ ID NOs. of the chimeric constant regions (CGama / CDelta)

Domain Name from the N-terminal to the C-terminal end and its SEQ ID NOs. complex and SEQ First or second Third Domain Domain domains listed in ID NOs. of the junction domain junction + hinge Dimensioning TCR (C1 or C2) chain: (CJ) (CJ ') (D) dg_Design_1 HC HCJ4 CGamma CJ'3G1 FcG1

SEQ ID SEQ ID NO: 234 SEQ ID NO: 117 SEQ ID NO: 113 SEQ ID NO: 121 NO: 302 dg_Design_1 LC LCJ4 CDelta SEQ ID NO: 233 SEQ ID NO: 119 SEQ ID NO: 310 dg_Design_2 HC HCJ5 CGamma CJ ' 3G1 FcG1

SEQ ID SEQ ID NO: 232 SEQ ID NO: 118 SEQ ID NO: 113 SEQ ID NO: 121 NO: 302 dg_Design_2 LC LCJ5 CDelta SEQ ID NO: 231 SEQ ID NO: 120 SEQ ID NO: 115 dg_Design_2_no HCJ5 CGamma (N65Q) CJ'3G1 FcG1 _Glyco HC

SEQ ID SEQ ID NO: 216 SEQ ID NO: 118 SEQ ID NO: 114 SEQ ID NO: 121 NO: 302 dg_Design_2_no LCJ5 CDelta (N16Q + N79Q) _Glyco LC SEQ ID NO: 215 SEQ ID NO: 120 SEQ ID NO: 116 dg_Design_2_hy CGamma (T12C) peCys1_no_Glyc HCJ5 CJ'3G1 FcG1 (N65Q) o HC

SEQ ID SEQ ID NO: 218 SEQ ID NO: 118 SEQ ID NO: 333 SEQ ID NO: 121 NO: 302 dg_Design_2_hy peCys1_no_Glyc LCJ5 CDelta (N16C) (N79Q) o LC SEQ ID NO: 217 SEQ ID NO: 120 SEQ ID NO: 120 SEQ ID NO: 120 SEQ ID NO: 120 SEQ ID NO: 120 : 325 dg_Design_2_hy CGamma (Q57C) peCys2_no_Glyc HCJ5 CJ'3G1 FcG1 (N65Q) o HC

SEQ ID SEQ ID NO: 220 SEQ ID NO: 118 SEQ ID NO: 334 SEQ ID NO: 121 NO: 302 dg_Design_2_hy CDelta (V50C) (N16Q peCys2_no_Glyc LCJ5 + N79Q) o LC SEQ ID NO: 219 SEQ ID NO: 120 SEQ ID NO: 326 dg_Design_2_hy CGamma (M62C) peCys3_no_Glyc HCJ5 CJ'3G1 FcG1 (N65Q) o HC

SEQ ID SEQ ID NO: 222 SEQ ID NO: 118 SEQ ID NO: 335 SEQ ID NO: 121 NO: 302 dg_Design_2_hy CDelta (D46C) (N16Q peCys3_no_Glyc LCJ5 + N79Q) o LC SEQ ID NO: 221 SEQ ID NO: 120 SEQ ID NO: 327 dg_Design_2_Cy CGamma (S17C) HCJ5 CJ'3G1 FcG1 s2_no_Glyco HC (N65Q)

SEQ ID SEQ ID NO: 224 SEQ ID NO: 118 SEQ ID NO: 336 SEQ ID NO: 121 NO: 302 dg_Design_2_Cy CDelta (F12C) (N16Q + LCJ5 s2_no_Glyco LC N79Q) SEQ ID NO: 223 SEQ ID NO: 120 SEQ ID NO: 328 dg_Design_2_Cy CGamma (F14C) HCJ5 CJ'3G1 FcG1 s1_no_Glyco HC (N65Q)

SEQ ID SEQ ID NO: 226 SEQ ID NO: 118 SEQ ID NO: 337 SEQ ID NO: 121 NO: 302 dg_Design_2_Cy CDelta (M14C) (N16Q LCJ5 s1_no_Glyco LC + N79Q)

Domain Name from the N-terminal to the C-terminal end and its SEQ ID NOs. complex and SEQ First or second Third Domain Domain domains listed in ID NOs. of the junction domain junction + hinge TCR dimerization (C1 or C2) chain: (CJ) (CJ ') (D) SEQ ID NO: 225 SEQ ID NO: 120 SEQ ID NO: 329 dg_Design_2_Cy CGamma (E20C) HCJ5 CJ'3G1 FcG1 s3_no_Glyco HC (N65Q)

SEQ ID SEQ ID NO: 227 SEQ ID NO: 118 SEQ ID NO: 338 SEQ ID NO: 121 NO: 302 dg_Design_2_Cy CDelta (F12C) (N16Q + LCJ5 s3_no_Glyco LC N79Q) SEQ ID NO: 223 SEQ ID NO: 120 SEQ ID NO: 328 dg_Design_2_Cy CGamma (A19C) HCJ5 CJ'3G1 FcG1 s4_no_Glyco HC (N65Q)

SEQ ID SEQ ID NO: 229 SEQ ID NO: 118 SEQ ID NO: 339 SEQ ID NO: 121 NO: 302 dg_Design_2_Cy CDelta (F87C) (N16Q + LCJ5 s4_no_Glyco LC N79Q) SEQ ID NO: 228 SEQ ID NO: 120 SEQ ID NO: 330 dg_Design_2_Cy CGamma (A19C) HCJ5 CJ'3G1 FcG1 s5_no_Glyco HC (N65Q)

SEQ ID SEQ ID NO: 229 SEQ ID NO: 118 SEQ ID NO: 339 SEQ ID NO: 121 NO: 302 dg_Design_2_Cy CDelta (E88C) (N16Q LCJ5 s5_no_Glyco LC + N79Q) SEQ ID NO: 230 SEQ ID NO: 120 SEQ ID NO: 331 dg_crossed_Desi HCJ6 CDelta CJ'4G1 FcG1 gn_1 HC

SEQ ID SEQ ID NO: 236 SEQ ID NO: 123 SEQ ID NO: 332 SEQ ID NO: 127 NO: 302 dg_crossed_Desi LCJ6 CGamma gn_1 LC SEQ ID NO: 235 SEQ ID NO: 125 SEQ ID NO: 340 dg_crossed_Desi HCJ7 CDelta CJ ' 4G1 FcG1 gn_2 HC

SEQ ID SEQ ID NO: 238 SEQ ID NO: 124 SEQ ID NO: 332 SEQ ID NO: 127 NO: 302 dg_crossed_Desi LCJ7 CGamma gn_2 LC SEQ ID NO: 237 SEQ ID NO: 126 SEQ ID NO: 340 TABLE 19. Sequences for examples of chimeric constant regions Drawings Models Type of SEQ ID Sequences CAlfa / CBeta Chain NO

KRTVAAPDPAVYQLRDSKSSDKSVCLFTDF LCJ1- DSQTNVSQSKDSDVYITDKCVLDMRSMDFK LC 176 CAlpha (T49C) SNSAVAWSNKSDFACANAFNNSIIPEDTFF

PSPESS design_1 SSASKNVFPPEVAVFEPSEAEISHTQKATL (IgG1) HCJ1- standard VCLATGFYPDHVELSWWVNGKEVHSGVCTD cbets PQPLKEQPALNDSRYALSSRLRVSATFWQN 177 HC (S56C) - PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ CJ'1G1- IVSAEAWGRASDKTHTCPPCPAPEAAGGPS Fc (G1) VFLFPPKPKDTLMISRTPEVTCVVVDVSHE

DPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF LCJ2- DSQTNVSQSKDSDVYITDKCVLDMRSMDFK LC 178 CAlpha (T49C) SNSAVAWSNKSDFACANAFNNSIIPEDTFF

PSPESS LEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTD

PQPLKEQPALNDSRYALSSRLRVSATFWQN Design_2 PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ (IgG1) HCJ2- standard IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets VFLFPPKPKDTLMISRTPEVTCVVVDVSHE 179 HC (S56C) - CJ'1G1- DPEVKFNWYVDGVEVHNAKTKPREEQYNST Fc (G1) YRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

KRTVAAPDPAVYQLRDSKSSDKSVCLFTDF LCJ1- DSQTNVSQSKDSDVYITDKCVLDMRSMDFK LC 176 CAlpha (T49C) SNSAVAWSNKSDFACANAFNNSIIPEDTFF

PSPESS SSASKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTD

PQPLKEQPALNDSRYALSSRLRVSATFWQN Design_5 PRNHFRCQVQFYPSNQIVSAEAWGRASDKT (IgG1) HCJ1- standard cbets HTCPPCPAPEAAGGPSVFLFPPKPKDTLMI (S56C) (FG- SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE HC 180 DE +) - VHNAKTKPREEQYNSTYRVVSVLTVLHQDW CJ'1G1- LNGKEYKCKVSNKALPAPIEKTISKAKGQP Fc (G1)

REPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF LCJ2- DSQTNVSQSKDSDVYITDKCVLDMRSMDFK LC 178 CAlpha (T49C) SNSAVAWSNKSDFACANAFNNSIIPEDTFF

PSPESS LEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTD

PQPLKEQPALNDSRYALSSRLRVSATFWQN Design_6 PRNHFRCQVQFYPSNQIVSAEAWGRASDKT (IgG1) HCJ2- standard cbets HTCPPCPAPEAAGGPSVFLFPPKPKDTLMI (S56C) (FG- 181 HC SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE +) - VHNAKTKPREEQYNSTYRVVSVLTVLHQDW CJ'1G1- LNGKEYKCKVSNKALPAPIEKTISKAKGQP Fc (G1)

REPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF LCJ2- DSQTNVSQSKDSDVYITDKCVLDMRSMDFK LC 178 CAlpha (T49C) SNSAVAWSNKSDFACANAFNNSIIPEDTFF

PSPESS LEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTD

PQPLKEQSGRYALSSRLRVSATFWQNPRNH Design_6a FRCQVQFYPSNQIVSAEAWGRASDKTHTCP (IgG1) HCJ2- standard cbets PCPAPEAAGGPSVFLFPPKPKDTLMISRTP (S56C) (FG- 182 HC EVTCVVVDVSHEDPEVKFNWYVDGVEVHNA +) - KTKPREEQYNSTYRVVSVLTVLHQDWLNGK CJ'1G1- EYKCKVSNKALPAPIEKTISKAKGQPREPQ Fc (G1)

VYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK

KLEDLKNVFPPEVAVFEPSEAEISHTQKAT LCJ3- LVCLATGFYPDHVELSWWVNGKEVHSGVCT LC 183 CBeta DPQPLKEQPALNDSRYALSSRLRVSATFWQ (S56C) NPRNHFRCQVQFYGLSENDEWTQDRA

QIVSAEAWGRA SSASIQNPDPAVYQLRDSKSSDKSVCLFTD

FDSQTNVSQSKDSDVYITDKCVLDMRSMDF Design_3 KSNSAVAWSNKSDFACANAFNNSIIPEDTF (IgG1) crossed HCJ2- FPSPESSSDKTHTCPPCPAPEAAGGPSVFL CBeta FPPKPKDTLMISRTPEV56 ()

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV CJ’1G1- VSVLTVLHQDWLNGKEYKCKVSNKALPAPI Fc (G1) EKTISKAKGQPREPQVYTLPPSREEMTKNQ

VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

KLEDLKNVFPPEVAVFEPSEAEISHTQKAT LCJ4- LVCLATGFYPDHVELSWWVNGKEVHSGVCT LC 183 CBeta DPQPLKEQPALNDSRYALSSRLRVSATFWQ (S56C) NPRNHFRCQVQFYGLSENDEWTQDRAK

QIVSAEAWGRA SSPDIQNPDPAVYQLRDSKSSDKSVCLFTD

FDSQTNVSQSKDSDVYITDKCVLDMRSMDF Design_4 KSNSAVAWSNKSDFACANAFNNSIIPEDTF (IgG1) crossed HCJ4-

FPSPESSSDKTHTCPPCPAPEAAGGPSVFL CAlpha FPPKPKDTLMISRTPEVTCVVVDVSHEDPE HC 185 (T49C) - VKFNWYVDGVEVHNAKTKPREEQYNSTYRV CJ’2G1- VSVLTVLHQKPREV

VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK LCJ3- KLEDLKNVFPPEVAVFEPSEAEISHTQKAT Design_7 LVCLATGFYPDHVELSWWVNGKEVHSGVCT CBeta (IgG1) LC 186 crossed (S56C) (FG- DPQPLKEQALN

SSASIQNPDPAVYQLRDSKSSDKSVCLFTD FDSQTNVSQSKDSDVYITDKCVLDMRSMDF

KSNSAVAWSNKSDFACANAFNNSIIPEDTF HCJ3-

FPSPESSSDKTHTCPPCPAPEAAGGPSVFL CAlpha FPPKPKDTLMISRTPEVTCVVVDVSHEDPE HC 184 (T49C) - VKFNWYVDGVEVHNAKTKPREEQYNSTYRV CJ’2G1- VSVLTVLHQKPREV

VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK LCJ4- KLEDLKNVFPPEVAVFEPSEAEISHTQKAT CBeta LVCLATGFYPDHVELSWWVNGKEVHSGVCT LC 186 (S56C) (FG- DPQPLKEQPALNDSRYALQRLNFRHFRFNFRHFRQFRQSFRQSFRQSFRQSRFRHVSFRFSF

SSPDIQNPDPAVYQLRDSKSSDKSVCLFTD

FDSQTNVSQSKDSDVYITDKCVLDMRSMDF Design_8 KSNSAVAWSNKSDFACANAFNNSIIPEDTF (IgG1) HCJ4-

FPSPESSSDKTHTCPPCPAPEAAGGPSVFL crossed CAlpha FPPKPKDTLMISRTPEVTCVVVDVSHEDPE HC 185 (T49C) - VKFNWYVDGVEVHNAKTKPREEQYNSTYRV CJ’2G1- VSVLTVLHQKR

VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK LCJ2- KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF CAlpha DSQTQVSQSKDSDVYITDKCVLDMRSMDFK LC 187 (T49C) (N34Q + N68

SNSAVAWSQKSDFACANAFQNSIIPEDTFF Q + N79Q) PSPESS

LEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTD

PQPLKEQPALQDSRYALSSRLRVSATFWQN Design_2- PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJ2- QQQQ (IgG1) IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets (S56C) VFLFPPKPKDTLMISRTPEVTCVVVDVSHE 188 HC (N69Q) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK LCJ2- KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF CAlpha DSQTAVSQSKDSDVYITDKCVLDMRSMDFK LC 189 (T49C) (N34A + N68

SNSAVAWSAKSDFACANAFANSIIPEDTFF A + N79A) PSPESS

LEDLKNVFPPEVAVFEPSEAEISHTQKATL Design_2- AAAA (IgG1) HCJ2- VCLATGFYPDHVELSWWVNGKEVHSGVCTD CBeta PQPLKEQPALADSRYALSSRLRVSATFWQN (S56C) HC 190 (N69A) -

PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ CJ’1G1- IVSAEAWGRASDKTHTCPPCPAPEAAGGPS Fc (G1) VFLFPPKPKDTLMISRTPEVTCVVVDVSHE

DPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK LCJ2- KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF CAlpha DSQTQVSQSKDSDVYITDKCVLDMRSMDFK LC 191 (T49C) (N34Q + N68

SNSAVAWSSKSDFACANAFKNSIIPEDTFF S + N79K) PSPESS

LEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTD

PQPLKEQPALEDSRYALSSRLRVSATFWQN Design_2- PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJ2- QSKE (IgG1) IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets (S56C) VFLFPPKPKDTLMISRTPEVTCVVVDVSHE 192 HC (N69E) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK LCJ2- KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF CAlpha DSQTAVSQSKDSDVYITDKCVLDMRSMDFK LC 193 (T49C) (N34A + N68

SNSAVAWSSKSDFACANAFKNSIIPEDTFF S + N79K) PSPESS

LEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTD

PQPLKEQPALEDSRYALSSRLRVSATFWQN Design_2- PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ ASKE (IgG1) HCJ2- IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets VFLFPPKPKDTLMISRTPEVTCVVVDVSHE 192 HC (S56C) (N69 E) -CJ'1G1- DPEVKFNWYVDGVEVHNAKTKPREEQYNST Fc (G1) YRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK LCJ2- KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF CAlpha DSQTQVSQSKDSDVYITDKCVLDMRSMDFK (T49C) LC 194 SQSAVAWSQKSDFACF (+49)

LEDLKNVFPPEVAVFEPSEAEISHTQKATL Design_2- VCLATGFYPDHVELSWWVNGKEVHSGVCTD

ANIMALS PQPLKEQPALQDSRYALSSRLRVSATFWQN HCJ2- (IgG1) PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ cbets (S56C) IVSAEAWGRASDKTHTCPPCPAPEAAGGPS 195 HC (N69Q) - VFLFPPKPKDTLMISRTPEVTCVVVDVSHE CJ'1G1- DPEVKFNWYVDGVEVHNAKTKPREEQYNST Fc (G1)

YRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK KPDIQNPDPAVYQLRDSKSSDKSVCLFTDF

DSQTQVSQSKDSDVYITDKCVLDMRSMDFK LC 187

SNSAVAWSQKSDFACANAFQNSIIPEDTFF PSPESS LEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTD

PQPLKEQPALQDSRYALSSRLRVSATFWQN Design_2- PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJ2- QQQQ (IgG4) cbets IVSAEAWGRYGPPCPPCPAPEFLGGPSVFL (S56C) FPPKPKDTLMISRTPEVTCVVVDVSQEDPE HC 196 (N69Q) - VQFNWYVDGVEVHNAKTKPREEQFNSTYRV CJ'1G4- VSVLTVLHQDWLNGKEYKCKVSNKGLPSSI Fc (G4)

EKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNV

FSCSVMHEALHNHYTQKSLSLSLGK SEQ ID Name Drawings Pre-Alpha- Beta String NO Strings

KPTGVGGTPFPSLAPPIMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY LC 197 CPreAlpha (N50Q) GPSPATDGTWTNLAHLSLPSEELASWEPLV

CHTGPGAEGHSRSTQPMHLSGEASTART LEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVSTD

PQPLKEQPALQDSRYALSSRLRVSATFWQN Design_1_Pr PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ e_TCR_Conj HCJB- unction'1 IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets VFLFPPKPKDTLMISRTPEVTCVVVDVSHE 198 HC (N69Q) - CJ'1G1- DPEVKFNWYVDGVEVHNAKTKPREEQYNST Fc (G1) YRVVSVLTVLHQDWLNGKEYKCKVSNKALP

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK LCJB- KPTGVGGTPFPSLAPPIMLLVDGKQQMVVV CPreAlpha CLVLDVAPPGLDSPIWFSAGQGSALDAFTC LC 199 (Y59C) GPSPATDGTWTNLAHLSLPSEASG

LEDLKNVFPPEVAVFEPSEAEISHTQKATL

VCLATGFYPDHVELSWWVNGKEVHSGVSTD Design_2_Pr PQPLKEQPALQDSRYALCSRLRVSATFWQN e_TCR_Conj PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJB- unction'1_Cys cbets IVSAEAWGRASDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHE 10 (S76C) 200 HC (N69Q) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

VL (CD3) - KPTGVGGTPFPSLCPPIMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY LC 201 CPreAlpha (A13C)

GPSPATDGTWTNLAHLSLPSEELASWEPLV (N50Q) CHTGPGAEGHSRSTQPMHLSGEASTART

LEDLKNVFPPEVAVCEPSEAEISHTQKATL

VCLATGFYPDHVELSWWVNGKEVHSGVSTD Design_3_Pr PQPLKEQPALQDSRYALSSRLRVSATFWQN e_TCR_Conj PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJB- unction'1_Cys IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets 11 VFLFPPKPKDTLMISRTPEVTCVVVDVSHE (F13C) 202 HC (N69Q) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - KPTGVGGTPFPSLCPPIMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY LC 201 CPreAlpha (A13C)

GPSPATDGTWTNLAHLSLPSEELASWEPLV (N50Q) CHTGPGAEGHSRSTQPMHLSGEASTART

LEDLKNVFPPEVAVFEPCEAEISHTQKATL

VCLATGFYPDHVELSWWVNGKEVHSGVSTD Design_4_Pr PQPLKEQPALQDSRYALSSRLRVSATFWQN e_TCR_Conj PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJB- unction'1_Cys IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets VFLFPPKPKDTLMISRTPEVTCVVVDVSHE 12 (S16C) 203 HC (N69Q) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - KPTGVGGTPFPCLAPPIMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY LC 204 CPreAlpha (S11C)

GPSPATDGTWTNLAHLSLPSEELASWEPLV (N50Q) CHTGPGAEGHSRSTQPMHLSGEASTART

LEDLKNVFPPEVAVFEPCEAEISHTQKATL

VCLATGFYPDHVELSWWVNGKEVHSGVSTD Design_5_Pr PQPLKEQPALQDSRYALSSRLRVSATFWQN e_TCR_Conj PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJB- unction'1_Cys IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets VFLFPPKPKDTLMISRTPEVTCVVVDVSHE 13 (S16C) 203 HC (N69Q) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK Design_6_Pr VL (CD3) - KPTGVGGTPFPCLAPPIMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY e_TCR_Conj LC 204 CPreAlpha un’1

GPSPATDGTWTNLAHLSLPSEELASWEPLV 14 CHTGPGAEGHSRSTQPMHLSGEASTART (N50Q)

LEDLKNVFPPEVAVFEPSECEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALQDSRYALSSRLRVSATFWQN HCJB-

PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ CBeta IVSAEAWGRASDKTHTCPPCPAPEAAGGPS (A18C) VFLFPPKPKDTLMISRTPEVTCVVVDVSHE HC 205 (N69Q) - DPEVKFNWYVDKVEHVKHVKVVVVVVVVVVVVQQQQQQQQKNKKVKVKVKQQQQQQQQQQQKKKKKKNKKKKKKKKKKKKKKKKKKKKKKKKKKKKNKKTKKKKKKKNKKTKKNKKTKKKKTKKKKNKKTKKNTKKTKKTKKKKNKKTKKNKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKGTKH

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - KPTGVGGTPFPCLAPPIMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY LC 204 CPreAlpha (S11C)

GPSPATDGTWTNLAHLSLPSEELASWEPLV (N50Q) CHTGPGAEGHSRSTQPMHLSGEASTART

LEDLKNVFPPEVAVFEPSEACISHTQKATL

VCLATGFYPDHVELSWWVNGKEVHSGVSTD Design_7_Pr PQPLKEQPALQDSRYALSSRLRVSATFWQN e_TCR_Conj PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJB- unction'1_Cys IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets 15 VFLFPPKPKDTLMISRTPEVTCVVVDVSHE (E19C) 206 HC (N69Q) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - KPTGVGGTPFPSLAPPIMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY LC 207 CPreAlpha (S62C)

GPCPATDGTWTNLAHLSLPSEELASWEPLV (N50Q) CHTGPGAEGHSRSTQPMHLSGEASTART

LEDLKNVFPPEVAVFEPSEAEISHTQKATL

VCLATGFYPDHVELSWWVNGKEVHSGVCTD Design_8_Pr PQPLKEQPALQDSRYALSSRLRVSATFWQN e_TCR_Conj PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJB- unction'1_Cys IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets 1_4L4T_1 VFLFPPKPKDTLMISRTPEVTCVVVDVSHE (S56C) 208 HC (N69Q) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - KPTGVGGTPFPSLAPPIMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY LC 209 CPreAlpha (T65C)

GPSPACDGTWTNLAHLSLPSEELASWEPLV Design_9_Pr CHTGPGAEGHSRSTQPMHLSGEASTART (N50Q) e_TCR_Conj unction’1_Cys HCJB- LEDLKNVFPPEVAVFEPSEAEISHTQKATL HCeta VCLATGKEVT4 (2) (2)

PQPLKEQPALQDSRYALSSRLRVSATFWQN CJ’1G1- PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ Fc (G1) IVSAEAWGRASDKTHTCPPCPAPEAAGGPS

VFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - KPTGVGGTPFPSLAPPCMLLVDGKQQMVVV LCJB- CLVLDVAPPGLDSPIWFSAGQGSALDAFTY LC 210 CPreAlpha (I16C)

GPSPATDGTWTNLAHLSLPSEELASWEPLV (N50Q) CHTGPGAEGHSRSTQPMHLSGEASTART

LEDLKNVFPPEVCVFEPSEAEISHTQKATL

VCLATGFYPDHVELSWWVNGKEVHSGVSTD Design_10_P PQPLKEQPALQDSRYALSSRLRVSATFWQN re_TCR_Conj PRNHFRCQVQFYGLSENDEWTQDRAKPVTQ HCJB- unction'1_Cys IVSAEAWGRASDKTHTCPPCPAPEAAGGPS cbets 4 VFLFPPKPKDTLMISRTPEVTCVVVDVSHE (A11C) 211 HC (N69Q) - DPEVKFNWYVDGVEVHNAKTKPREEQYNST CJ'1G1- YRVVSVLTVLHQDWLNGKEYKCKVSNKALP Fc (G1)

APIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - KLEDLKNVFPPEVAVFEPCEAEISHTQKAT HCJB- LVCLATGFYPDHVELSWWVNGKEVHSGVST CBeta Leve 212 (N69Q,

DPQPLKEQPALQDSRYALSSRLRVSATFWQ S16C) - NPRNHFRCQVQFYGLSENDEWTQDRAKPVT CJ’1G QIVSAEAWGRA

SSASGVGGTPFPCLAPPIMLLVDGKQQMVV VCLVLDVAPPGLDSPIWFSAGQGSALDAFT

YGPSPATDGTWTNLAHLSLPSEELASWEPL Pre- TCR_Design_

Heavy VCHTGPGAEGHSRSTQPMHLSGEASTARTS 5_crossed_1 HCJC- DKTHTCPPCPAPEAAGGPSVFLFPPKPKDT CPreAlpha LMISRTPEVTCVVVDVSHEDPEVKFNWYVD (more junction 213 (S11C, GVEVHNAKTKPREEQYNSTYRVVSVLTVLH antibody) N50Q) - Fc QDWLNGKEYKCKVSNKALPAPIEKTISKAK CJ'2G1-

GQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK VL (CD3) - Take KLEDLKNVFPPEVAVFEPCEAEISHTQKAT HCJB- LVCLATGFYPDHVELSWWVNGKEVHSGVST cbets DPQPLKEQPALQDSRYALSSRLRVSATFWQ 212 (N69Q, S16C) - NPRNHFRCQVQFYGLSENDEWTQDRAKPVT CJ'1G QIVSAEAWGRA Pre SSPTGVGGTPFPCLAPPIMLLVDGKQQMVV TCR_Design_ 5_crossed_2 VCLVLDVAPPGLDSPIWFSAGQGSALDAFT HCJD- YGPSPATDGTWTNLAHLSLPSEELASWEPL Heavy CPreAlpha (Binding more VCHTGPGAEGHSRSTQPMHLSGEASTARTS 214 (S11) - Pre-TCR) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDT CJ'2G1- Fc

LMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK VL (CD3) -

KLEDLKNVFPPEVAVFEPSECEISHTQKAT LCJC- LVCLATGFYPDHVELSWWVNGKEVHSGVST Light 215 CBeta DPQPLKEQPALQDSRYALSSRLRVSATFWQ Pre- (A18C, NPRNHFRCQVQQYRARAQ_CRT_CREDIT_TEMPLE) (6)

KLEDLKNVFPPEVAVFEPSECEISHTQKAT

Take LVCLATGFYPDHVELSWWVNGKEVHSGVST 215 DPQPLKEQPALQDSRYALSSRLRVSATFWQ Pre TCR_Design_ NPRNHFRCQVQFYGLSENDEWTQDRAKPVT 6_crossed_2 QIVSAEAWGRA Heavy (214 Binding more TCR) Delta- Drawings Name of SEQ ID NO Gamma Chain Sequences VL (CD3) - LC 215 EPRSQPHTKPSVFVMKQGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFDPAIVISPSGKYNA CDelta (N79 N16Q +

VKLGKYEDSNSVTCSVQHDQKTVHSTDFE Q) TDKQLDADVSPKPTIFLPSIAETKLQKAGT YLCLLEKFFPDVIKIHWQEKKSNTILGSQE

GNTMKTQDTYMKFSWLTVPEESLDKEHRCI dg_Design_2 VRHENNKNGVDQEIIFPPIKSDKTHTCPPC _no_Glyco HCJ5- PAPEAAGGPSVFLFPPKPKDTLMISRTPEV CGamma- TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT HC 216 CJ'3G1- Fc (G1) KPREEQYNSTYRVVSVLTVLHQDWLNGKEY (N65Q) KCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK VL (CD3) - EPRSQPHTKPSVFVMKCGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFDPAIVISPSGKYNA LC 217

VKLGKYEDSNSVTCSVQHDQKTVHSTDFE (N16C) (N79Q)

TDKQLDADVSPKPCIFLPSIAETKLQKAGT

YLCLLEKFFPDVIKIHWQEKKSNTILGSQE dg_Design_2 _hypeCys1_n

GNTMKTQDTYMKFSWLTVPEESLDKEHRCI HCJ5- o_Glyco Cgamma VRHENNKNGVDQEIIFPPIKSDKTHTCPPC (T12C) PAPEAAGGPSVFLFPPKPKDTLMISRTPEV 218 HC (N65Q) - TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT CJ'3G1- KPREEQYNSTYRVVSVLTVLHQDWLNGKEY Fc (G1)

KCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK VL (CD3) - EPRSQPHTKPSVFVMKQGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFDPAICISPSGKYNA CDelta LC 219 (V50C)

VKLGKYEDSNSVTCSVQHDQKTVHSTDFE (N16Q + N79Q)

TDKQLDADVSPKPTIFLPSIAETKLQKAGT

YLCLLEKFFPDVIKIHWQEKKSNTILGSCE dg_Design_2 GNTMKTQDTYMKFSWLTVPEESLDKEHRCI _hypeCys2_n VRHENNKNGVDQEIIFPPIKSDKTHTCPPC HCJ5- o_Glyco PAPEAAGGPSVFLFPPKPKDTLMISRTPEV Cgamma (Q57C) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 220 HC (N65Q) - KPREEQYNSTYRVVSVLTVLHQDWLNGKEY CJ'3G1- KCKVSNKALPAPIEKTISKAKGQPREPQVY Fc (G1)

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK VL (CD3) - EPRSQPHTKPSVFVMKQGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFCPAIVISPSGKYNA CDelta LC 221 (D46C)

VKLGKYEDSNSVTCSVQHDQKTVHSTDFE (N16Q + N79Q)

TDKQLDADVSPKPTIFLPSIAETKLQKAGT

YLCLLEKFFPDVIKIHWQEKKSNTILGSQE dg_Design_2 GNTCKTQDTYMKFSWLTVPEESLDKEHRCI _hypeCys3_n VRHENNKNGVDQEIIFPPIKSDKTHTCPPC HCJ5- o_Glyco PAPEAAGGPSVFLFPPKPKDTLMISRTPEV Cgamma (M62C) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 222 HC (N65Q) - KPREEQYNSTYRVVSVLTVLHQDWLNGKEY CJ'3G1- KCKVSNKALPAPIEKTISKAKGQPREPQVY Fc (G1)

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK VL (CD3) - EPRSQPHTKPSVCVMKQGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFDPAIVISPSGKYNA CDelta LC 223 (F12C)

VKLGKYEDSNSVTCSVQHDQKTVHSTDFE (N16Q + N79Q)

TDKQLDADVSPKPTIFLPCIAETKLQKAGT

YLCLLEKFFPDVIKIHWQEKKSNTILGSQE dg_Design_2 GNTMKTQDTYMKFSWLTVPEESLDKEHRCI _Cys2_no_Gl VRHENNKNGVDQEIIFPPIKSDKTHTCPPC HCJ5- YCO PAPEAAGGPSVFLFPPKPKDTLMISRTPEV Cgamma (S17C) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 224 HC (N65Q) - KPREEQYNSTYRVVSVLTVLHQDWLNGKEY CJ'3G1- KCKVSNKALPAPIEKTISKAKGQPREPQVY Fc (G1)

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

VL (CD3) - EPRSQPHTKPSVFVCKQGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFDPAIVISPSGKYNA CDelta LC 225 (M14C)

VKLGKYEDSNSVTCSVQHDQKTVHSTDFE (N16Q + N79Q)

TDKQLDADVSPKPTICLPSIAETKLQKAGT

YLCLLEKFFPDVIKIHWQEKKSNTILGSQE dg_Design_2 GNTMKTQDTYMKFSWLTVPEESLDKEHRCI _Cys1_no_Gl VRHENNKNGVDQEIIFPPIKSDKTHTCPPC HCJ5- YCO PAPEAAGGPSVFLFPPKPKDTLMISRTPEV Cgamma (F14C) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 226 HC (N65Q) - KPREEQYNSTYRVVSVLTVLHQDWLNGKEY CJ'3G1- KCKVSNKALPAPIEKTISKAKGQPREPQVY Fc (G1)

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK VL (CD3) - EPRSQPHTKPSVCVMKQGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFDPAIVISPSGKYNA CDelta LC 223 (F12C)

VKLGKYEDSNSVTCSVQHDQKTVHSTDFE (N16Q + N79Q)

TDKQLDADVSPKPTIFLPSIACTKLQKAGT

YLCLLEKFFPDVIKIHWQEKKSNTILGSQE dg_Design_2 GNTMKTQDTYMKFSWLTVPEESLDKEHRCI _Cys3_no_Gl VRHENNKNGVDQEIIFPPIKSDKTHTCPPC HCJ5- YCO PAPEAAGGPSVFLFPPKPKDTLMISRTPEV Cgamma (E20C) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 227 HC (N65Q) - KPREEQYNSTYRVVSVLTVLHQDWLNGKEY CJ'3G1- KCKVSNKALPAPIEKTISKAKGQPREPQVY Fc (G1)

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK VL (CD3) - EPRSQPHTKPSVFVMKQGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFDPAIVISPSGKYNA CDelta LC 228 VKLGKYEDSNSVTCSVQHDQKTVHSTDCE (F87C) (N87 +)

TDKQLDADVSPKPTIFLPSICETKLQKAGT

YLCLLEKFFPDVIKIHWQEKKSNTILGSQE dg_Design_2 GNTMKTQDTYMKFSWLTVPEESLDKEHRCI _Cys4_no_Gl VRHENNKNGVDQEIIFPPIKSDKTHTCPPC HCJ5- YCO PAPEAAGGPSVFLFPPKPKDTLMISRTPEV Cgamma (A19C) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 229 HC (N65Q) - KPREEQYNSTYRVVSVLTVLHQDWLNGKEY CJ'3G1- KCKVSNKALPAPIEKTISKAKGQPREPQVY Fc (G1)

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

VL (CD3) - EPRSQPHTKPSVFVMKQGTNVACLVKEFYP LCJ5- KDIRINLVSSKKITEFDPAIVISPSGKYNA CDelta LC 230 (E88C)

VKLGKYEDSNSVTCSVQHDQKTVHSTDFC (N16Q + N79Q)

TDKQLDADVSPKPTIFLPSICETKLQKAGT

YLCLLEKFFPDVIKIHWQEKKSNTILGSQE dg_Design_2 GNTMKTQDTYMKFSWLTVPEESLDKEHRCI _Cys5_no_Gl VRHENNKNGVDQEIIFPPIKSDKTHTCPPC HCJ5- YCO PAPEAAGGPSVFLFPPKPKDTLMISRTPEV Cgamma (A19C) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT 229 HC (N65Q) - KPREEQYNSTYRVVSVLTVLHQDWLNGKEY CJ'3G1- KCKVSNKALPAPIEKTISKAKGQPREPQVY Fc (G1)

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK VL (CD3) - EPRSQPHTKPSVFVMKNGTNVACLVKEFYP LC 231 LCJ5- KDIRINLVSSKKITEFDPAIVISPSGKYNA CDelta VKLGKYEDSNSVTCSVQHDNKTVHSTDFEFE

TDKQLDADVSPKPTIFLPSIAETKLQKAGT YLCLLEKFFPDVIKIHWQEKKSNTILGSQE GNTMKTNDTYMKFSWLTVPEESLDKEHRCI

VRHENNKNGVDQEIIFPPIKSDKTHTCPPC dg_Design_2 HCJ5- PAPEAAGGPSVFLFPPKPKDTLMISRTPEV CGamma- TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT HC 232 CJ'3G1- KPREEQYNSTYRVVSVLTVLHQDWLNGKEY Fc (G1) KCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK VL (CD3) - KPRSQPHTKPSVFVMKNGTNVACLVKEFYP LC 233 LCJ4- KDIRINLVSSKKITEFDPAIVISPSGKYNA CDelta VKLGKYEDSNSVTCSVQHDNKTVHSTDFEFE

SSASLDADVSPKPTIFLPSIAETKLQKAGT YLCLLEKFFPDVIKIHWQEKKSNTILGSQE GNTMKTNDTYMKFSWLTVPEESLDKEHRCI

VRHENNKNGVDQEIIFPPIKSDKTHTCPPC dg_Design_1 HCJ4- PAPEAAGGPSVFLFPPKPKDTLMISRTPEV CGamma- TCVVVDVSHEDPEVKFNWYVDGVEVHNAKT HC 234 CJ’3G1- KPREEKY

TLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

KDKQLDADVSPKPTIFLPSIAETKLQKAGT VL (CD3) - YLCLLEKFFPDVIKIHWQEKKSNTILGSCE LC 235 LCJ6- dg_crossed_ CGamma

GNTMKTQDTYMKFSWLTVPEESLDKEHRCI Design_1 VRHENNKNGVDQEIIFPPIKTDVITMD HCJ6-

SSRSQPHTKPSVFVMKQGTNVACLVKEFYP HC 236 KDIRINLVSSKKITEFDPAICISPSGKYNA CDelta-

CJ’4G1- VKLGKYEDSNSVTCSVQHDQKTVHSTDEPK Fc (G1) SCDKTHTCPPCPAPEAAGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

KDKQLDADVSPKPTIFLPSIAETKLQKAGT VL (CD3) - YLCLLEKFFPDVIKIHWQEKKSNTILGSCE LC 237 LCJ7- CGamma GNTMKTQDTYMKFSWLTVPEESLDKEHRCI

VRHENNKNGVDQEIIFPPIKTDVITMD EPRSQPHTKPSVFVMKQGTNVACLVKEFYP KDIRINLVSSKKITEFDPAICISPSGKYNA

VKLGKYEDSNSVTCSVQHDQKTVHSTDEPK dg_crossed_ Design_2

SCDKTHTCPPCPAPEAAGGPSVFLFPPKPK HCJ7- DTLMISRTPEVTCVVVDVSHEDPEVKFNWY CDelta- HC 238 VDGVEVHNAKTKPREEQYNSTYRVVSVLTV CJ’4G1- FC (G1) LHQKLK

AKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK TABLE 20. Sequences for the examples of peptide complexes Drawings SEQ Name ID Sequences Alpha-Beta String No

DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJ1- LC 1 CAlpha

VYYCTQSHTLRTFGGGTKVEIKRTVAAPDP (T49C) AVYQLRDSKSSDKSVCLFTDFDSQTNVSQS

KDSDVYITDKCVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY

NENFKGRVTITADKSTSTAYMELSSLRSED T3-design_1 TAVYYCARDGYSLYYFDYWGQGTLVTVSSA (IgG1) Normal SKNVFPPEVAVFEPSEAEISHTQKATLVCL ATGFYPDHVELSWWVNGKEVHSGVCTDPQP VH (CD3) - CH 2 LKEQPALNDSRYALSSRLRVSATFWQNPRN HCJ1- HFRCQVQFYGLSENDEWTQDRAKPVTQIVS cbets (C S56) -CJ'1G1- AEAWGRASDKTHTCPPCPAPEAAGGPSVFL Fc (G1) FPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK T3-Design_2 VL (CD3) - DIVMTQSPDSLAVSLGERATINCKSSQSLL LC 3 (IgG1) LCJ2- NSRTRKNYLAWYQQKPGQPPKLLIYWASTR normal CAlpha QSGVTTGGT

AVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVLED LKNVFPPEVAVFEPSEAEISHTQKATLVCL

ATGFYPDHVELSWWVNGKEVHSGVCTDPQP VH (CD3) - LKEQPALNDSRYALSSRLRVSATFWQNPRN HCJ2- HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 4 CBeta (S56 C) -CJ’SCPKPHTTDHTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTLXLXTXTXTTTTTTTTTTTWWTWTTTTTGTFFGTWFVFVFP

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJ1- LC 1 VYYCTQSHTLRTFGGGTKVEIKRTVAAPDP CAlpha (T49C) AVYQLRDSKSD

KDSDVYITDKCVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVSSA T3-Design_5 (IgG1)

Normal SKNVFPPEVAVFEPSEAEISHTQKATLVCL VH (CD3) - ATGFYPDHVELSWWVNGKEVHSGVCTDPQP HCJ1- LKEQPALNDSRYALSSRLRVSATFWQNPRN cbets (S56 HFRCQVQFYPSNQIVSAEAWGRASDKTHTC C HC 5) (DE FG- +) - PPCPAPEAAGGPSVFLFPPKPKDTLMISRT CJ'1G1- PEVTCVVVDVSHEDPEVKFNWYVDGVEVHN Fc (G1) AKTKPREEQYNSTYRVVSVLTVLHQDWLNG

KEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJ2- LC 3 CAlpha

VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP T3-Design_6 AVYQLRDSKSSDKSVCLFTDFDSQTNVSQS (T49C) (IgG1) KDSDVYITDKCVLDMRSMDFKSNSAVAWSN normal

KSDFACANAFNNSIIPEDTFFPSPESS VH (CD3) - QVQLVQSGAEVKKPGSSVKVSCKASGFAFT HC 6 HCJ2- DYYIHWVRQAPGQGLEWMGWISPGNVNTKY CBeta (S56

C) (FG- NENFKGRVTITADKSTSTAYMELSSLRSED DE +) - TAVYYCARDGYSLYYFDYWGQGTLVTVLED CJ’1G1- Fc (G1)

LKNVFPPEVAVFEPSEAEISHTQKATLVCL ATGFYPDHVELSWWVNGKEVHSGVCTDPQP LKEQPALNDSRYALSSRLRVSATFWQNPRN HFRCQVQFYPSNQIVSAEAWGRASDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJ2- LC 3 VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP CAYPH (T49C) AVYQL

KDSDVYITDKCVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED T3-Design_6a LKNVFPPEVAVFEPSEAEISHTQKATLVCL (IgG1) Normal ATGFYPDHVELSWWVNGKEVHSGVCTDPQP VH (CD3) - LKEQ - SG-- HCJ2- RYALSSRLRVSATFWQNPRNHFRCQVQFYP cbets (S56 HC 7 SNQIVSAEAWGRASDKTHTCPPCPAPEAAG C) (FG-A -) - Fc CJ'1G1- GPSVFLFPPKPKDTLMISRTPEVTCVVVDV (G1) SHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG K DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA VL (CD3) - VYYCTQSHTLRTFGGGTKVEIKLEDLKNVF LCJ3- LC 8 CBeta (S56

PPEVAVFEPSEAEISHTQKATLVCLATGFY C) PDHVELSWWVNGKEVHSGVCTDPQPLKEQP

ALNDSRYALSSRLRVSATFWQNPRNHFRCQ T3-Design_3 VQFYGLSENDEWTQDRAKPVTQIVSAEAWG (IgG1) crossed RA

QVQLVQSGAEVKKPGSSVKVSCKASGFAFT VH (CD3) - HCJ3- DYYIHWVRQAPGQGLEWMGWISPGNVNTKY CAlpha NENFKGRVTITADKSTSTAYMELSSLRSED HC 9 (T49C) - TAVYYDS_GS

QTNVSQSKDSDVYITDKCVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPS PESSSDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA VL (CD3) - VYYCTQSHTLRTFGGGTKVEIKLEDLKNVF LCJ4- LC 8 PPEVAVFEPSEAEISHTQKATLVCLATGFY CBeta (S56 C) PDHCTQPL

ALNDSRYALSSRLRVSATFWQNPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWG FROG QVQLVQSGAEVKKPGSSVKVSCKASGFAFT

DYYIHWVRQAPGQGLEWMGWISPGNVNTKY T3-Design_4 NENFKGRVTITADKSTSTAYMELSSLRSED (IgG1) TAVYYCARDGYSLYYFDYWGQGTLVTVSSP crossed DIQNPDPAVYQLRDSKSSDKSVCLFTDFDS VH (CD3) - QTNVSQSKDSDVYITDKCVLDMRSMDFKSN HCJ4- SAVAWSNKSDFACANAFNNSIIPEDTFFPS CAlpha PESSSDKTHTCPPCPAPEAAGGPSVFLFPP HC 10 (T49C) - CJ'2G1- KPKDTLMISRTPEVTCVVVDVSHEDPEVKF Fc (G1) NWYVDGVEVHNAKTKPREEQYNSTYRVVSV

LTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJ3- VYYCTQSHTLRTFGGGTKVEIKLEDLKNVF LC 11 CBeta (S56 PPEVVTNV)

ALNDSRYALSSRLRVSATFWQNPRNHFRCQ VQFYPSNQIVSAEAWGRA

QVQLVQSGAEVKKPGSSVKVSCKASGFAFT T3-Design_7 DYYIHWVRQAPGQGLEWMGWISPGNVNTKY (IgG1) NENFKGRVTITADKSTSTAYMELSSLRSED crossed TAVYYCARDGYSLYYFDYWGQGTLVTVSSA VH (CD3) - HCJ3- SIQNPDPAVYQLRDSKSSDKSVCLFTDFDS CAlpha 9 QTNVSQSKDSDVYITDKCVLDMRSMDFKSN HC (T49C) - SAVAWSNKSDFACANAFNNSIIPEDTFFPS CJ'2G1- PESSSDKTHTCPPCPAPEAAGGPSVFLFPP Fc (G1)

KPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJ4- VYYCTQSHTLRTFGGGTKVEIKLEDLKNVF LC 11 CBeta (S56 PPEVVTNV)

ALNDSRYALSSRLRVSATFWQNPRNHFRCQ VQFYPSNQIVSAEAWGRA QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY

NENFKGRVTITADKSTSTAYMELSSLRSED T3-Design_8 TAVYYCARDGYSLYYFDYWGQGTLVTVSSP (IgG1) DIQNPDPAVYQLRDSKSSDKSVCLFTDFDS crossed VH (CD3) - QTNVSQSKDSDVYITDKCVLDMRSMDFKSN HCJ4- SAVAWSNKSDFACANAFNNSIIPEDTFFPS CAlpha PESSSDKTHTCPPCPAPEAAGGPSVFLFPP HC 10 (T49C) - CJ'2G1- KPKDTLMISRTPEVTCVVVDVSHEDPEVKF Fc (G1) NWYVDGVEVHNAKTKPREEQYNSTYRVVSV

LTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ2- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CAlpha LC 12 (T49C)

VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP (N34Q + N68 AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS Q + N79Q) KDSDVYITDKCVLDMRSMDFKSNSAVAWSQ

KSDFACANAFQNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED T3-Design_2- LKNVFPPEVAVFEPSEAEISHTQKATLVCL QQQQ (IgG1) ATGFYPDHVELSWWVNGKEVHSGVCTDPQP VH (CD3) - HCJ2- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (S56 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 13C) (N69Q) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - DIVMTQSPDSLAVSLGERATINCKSSQSLL LCJ2- NSRTRKNYLAWYQQKPGQPPKLLIYWASTR T3-Design_2- LC 14 AAAA (IgGTGGT)

(N34A + N68 AVYQLRDSKSSDKSVCLFTDFDSQTAVSQS A + N79A) KDSDVYITDKCVLDMRSMDFKSNSAVAWSA

KSDFACANAFANSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVLED LKNVFPPEVAVFEPSEAEISHTQKATLVCL

ATGFYPDHVELSWWVNGKEVHSGVCTDPQP VH (CD3) - HCJ2- LKEQPALADSRYALSSRLRVSATFWQNPRN CBeta (S56 HFRCQVQFYGLSENDEWTQDRAKPVTQVSQTTGPDHVTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTX mot mot_x

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ2- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CAlpha LC 16 (T49C) (

VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP N34Q + N68 AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS S + N79K) KDSDVYITDKCVLDMRSMDFKSNSAVAWSS

KSDFACANAFKNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED T3-Design_2- LKNVFPPEVAVFEPSEAEISHTQKATLVCL QSKE (IgG1) ATGFYPDHVELSWWVNGKEVHSGVCTDPQP VH (CD3) - HCJ2- LKEQPALEDSRYALSSRLRVSATFWQNPRN cbets (S56 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 17 C) (N69E) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ2- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CAlpha LC 18 (T49C) (

N34A + N68 VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP AVYQLRDSKSSDKSVCLFTDFDSQTAVSQS T3-Design_2- S + N79K) KDSDVYITDKCVLDMRSMDFKSNSAVAWSS ASKE (IgG1) KSDFACANAFKNSIIPEDTFFPSPESS VH (CD3) - QVQLVQSGAEVKKPGSSVKVSCKASGFAFT HCJ2- DYYIHWVRQAPGQGLEWMGWISPGNVNTKY cbets HC 17 (S56 NENFKGRVTITADKSTSTAYMELSSLRSED C) (N69E) - TAVYYCARDGYSLYYFDYWGQGTLVTVLED

CJ’1G1- LKNVFPPEVAVFEPSEAEISHTQKATLVCL Fc (G1) ATGFYPDHVELSWWVNGKEVHSGVCTDPQP

LKEQPALEDSRYALSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRASDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - LCJ2-

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR CAlpha QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LC 19 (T49C) VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP (N34Q + QQQQSQSDDSKDQSQDQSQDQSKDQSKDQSKDKDKDKDKDKDDDKKDKDKDDKDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDSKS

KSDFACANAFQNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED T3-Design_2-

QQQQQ

LKNVFPPEVAVFEPSEAEISHTQKATLVCL (IgG1) ATGFYPDHVELSWWVNGKEVHSGVCTDPQP VH (CD3) - HCJ2- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (S56 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 20 C) (N69Q) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LC 12 VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP

AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSQ KSDFACANAFQNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT

DYYIHWVRQAPGQGLEWMGWISPGNVNTKY T3-Design_2- QQQQ (IgG4) NENFKGRVTITADKSTSTAYMELSSLRSED VH (CD3) - TAVYYCARDGYSLYYFDYWGQGTLVTVLED HCJE- (LYCH)

ATGFYPDHVELSWWVNGKEVHSGVCTDPQP CJ’1G4- LKEQPALQDSRYALSSRLRVSATFWQNPRN Fc (G4) HFRCQVQFYGLSENDEWTQDRAKPVTQIVS

AEAWGRYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ2- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CAlpha T3 LC-12 VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP (T49C) (AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS N34Q + N68 Q + N79Q) KDSDVYITDKCVLDMRSMDFKSNSAVAWSQ

KSDFACANAFQNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVLED

LKNVFPPEVAVFEPSEAEISHTQKATLVCL VH (CD3) - ATGFYPDHVELSWWVNGKEVHSGVCTDPQP HCJ2- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (S56 HC-22 T3 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS C) (N69Q) - CJ'1G1- AEAWGRASDKTHTCPPCPAPEAAGGPSVFL Fc (G1) (Knowlton FPPKPKDTLMISRTPEVTCVVVDVSHEDPE b) VKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPCREEMTKNQ VSLWCLVKGFYPSDIAVEWESNGQPENNYK

TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV E17- FSCSVMHEALHNHYTQKSLSLSPGK Design_2- DIQLTQSPSFLSASVGDRVTITCSASSTVN QQQQ (IgG1) YMHWYQQKPGKAPLLL

FSGSGSGTEFTLTISSLQPEDFATYYCHQW VL (CD19) - SSYPYTFGQGTKLEIKRTVAAPSVFIFPPS U4-LC 23

CL DEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC QMQLVQSGPEVKKPGTSVKVSCKASGYAFT SYNMYWVRQARGQRLEWIGYIDPYNGDTTY NQKFKGRVTITRDMSTSTAYMELSSLRSED TAVYYCLTTAYAMDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYS VH (CD19) - LSSVVTVPSSSLGTQTYICNVNHKPSNTKV U4-HC 24 CH1-Fc (G1) DKRVEPKSCDKTHTCPPCPAPEAAGGPSVF (Hole) LFPPVVPTV

EVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVCTLPPSREEMTKN QVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLVSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) - DIVMTQSPDSLAVSLGERATINCKSSQSLL T3-LC 12 LCJ2- NSRTRKNYLAWYQQKPGQPPKLLIYWASTR CAlpha

E17- (T49C) QSGVPDRFSGSGSGTDFTLTISSLQAEDVA Design_2- (N34Q + N68 VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP QQQQ (IgG4) Q + N79Q)

AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSQ KSDFACANAFQNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVLED

LKNVFPPEVAVFEPSEAEISHTQKATLVCL VH (CD3) - ATGFYPDHVELSWWVNGKEVHSGVCTDPQP HCJ2- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (S56 HC-25 T3 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS C) (N69Q) - CJ'1G4- AEAWGRYGPPCPPCPAPEFLGGPSVFLFPP Fc (G4) KPKDTLMISRTPEVTCVVVDVSQEDPEVQF (Knob) NWYVDGVEVHNAKTKPREEQFNSTYRVVSV

LTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPCQEEMTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK DIQLTQSPSFLSASVGDRVTITCSASSTVN YMHWYQQKPGKAPKLLIYSTSNLASGVPSR

FSGSGSGTEFTLTISSLQPEDFATYYCHQW VL (CD19) - SSYPYTFGQGTKLEIKRTVAAPSVFIFPPS U4-LC 23

CL DEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC QMQLVQSGPEVKKPGTSVKVSCKASGYAFT SYNMYWVRQARGQRLEWIGYIDPYNGDTTY NQKFKGRVTITRDMSTSTAYMELSSLRSED TAVYYCLTTAYAMDYWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYS VH (CD19) - LSSVVTVPSSSLGTKTYTCNVDHKPSNTKV CH1- U4-HC 26 DKRVESKYGPPCPPCPAPEFLGGPSVFLFP Fc (G4) (Hole) PKPVVDTTM

FNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVS LSCAVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLVSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA T3-LC 12 VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP F16- AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS Design_2- QQQQ (IgG4) KDSDVY

KSDFACANAFQNSIIPEDTFFPSPESS

QVQLVQSGAEVKKPGSSVKVSCKASGFAFT T3-HC 25

DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVLED LKNVFPPEVAVFEPSEAEISHTQKATLVCL ATGFYPDHVELSWWVNGKEVHSGVCTDPQP LKEQPALQDSRYALSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPCQEEMTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK DIQLTQSPSFLSASVGDRVTITCSASSTVN YMHWYQQKPGKAPKLLIYSTSNLASGVPSR

FSGSGSGTEFTLTISSLQPEDFATYYCHQW VL (CD19) - SSYPYTFGQGTKLEIKRTVAAPSVFIFPPS U4-LC 23

CL DEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC QMQLVQSGPEVKKPGTSVKVSCKASGYAFT SYNMYWVRQARGQRLEWIGYIDPYNGDTTY NQKFKGRVTITRDMSTSTAYMELSSLRSED TAVYYCLTTAYAMDYWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVGGGGSGGGGSQMQLVQSGPEVKKPGT

SVKVSCKASGYAFTSYNMYWVRQARGQRLE VH (CD19) - CH1-

WIGYIDPYNGDTTYNQKFKGRVTITRDMST Spacer- STAYMELSSLRSEDTAVYYCLTTAYAMDYW U4-HC 27 VH (CD19) - GQGTLVTVSSASTKGPSVFPLAPCSRSTSE CH1- STAALGCLVKDYFPETLVSVTLSVLVLVLVLVLVLVLQTTLVLVLVLVLVLVLVSKVTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTF

YTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVCT LPPSQEEMTKNQVSLSCAVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLVSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK SVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKAL Fc-IgG1 (knob) 304 PAPIEKTISKAKGQPREPQVYTLPPCREEM

TKNQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TYRVVSVLTVLHQDWLNGKEYKCKVSNKGL Fc-IgG4 (knob) 305 PSSIEKTISKAKGQPREPQVYTLPPCQEEM

TKNQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJ2- LC 3 CAlpha

VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP (T49C) AVYQLRDSKSSDKSVCLFTDFDSQTNVSQS

KDSDVYITDKCVLDMRSMDFKSNSAVAWSN

KSDFACANAFNNSIIPEDTFFPSPESS T3-1 Fab QVQLVQSGAEVKKPGSSVKVSCKASGFAFT Design_2.his DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED VH (CD3) - HCJ2- TAVYYCARDGYSLYYFDYWGQGTLVTVLED cbets HC 28 (S56 LKNVFPPEVAVFEPSEAEISHTQKATLVCL C) on Conjuncti ATGFYPDHVELSWWVNGKEVHSGVCTDPQP 'LKEQPALNDSRYALSSRLRVSATFWQNPRN

HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGR DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LC 3 VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP

AVYQLRDSKSSDKSVCLFTDFDSQTNVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSN

KSDFACANAFNNSIIPEDTFFPSPESS T3 Fab-QVQLVQSGAEVKKPGSSVKVSCKASGFAFT Design_2.his DYYIHWVRQAPGQGLEWMGWISPGNVNTKY 2 NENFKGRVTITADKSTSTAYMELSSLRSED VH (CD3) - HCJ2- TAVYYCARDGYSLYYFDYWGQGTLVTVLED cbets HC 29 (S56 LKNVFPPEVAVFEPSEAEISHTQKATLVCL C) on Conjuncti ATGFYPDHVELSWWVNGKEVHSGVCTDPQP 'LKEQPALNDSRYALSSRLRVSATFWQNPRN

HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRAD

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ2- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CAlpha LC 12 (T49C)

VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP (AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS N34Q + N68 Q + N79Q) KDSDVYITDKCVLDMRSMDFKSNSAVAWSQ T3 Fab-KSDFACANAFQNSIIPEDTFFPSPESS Design_2- QVQLVQSGAEVKKPGSSVKVSCKASGFAFT QQQQ.his1 DYYIHWVRQAPGQGLEWMGWISPGNVNTKY VH (CD3) - HCJ2- NENFKGRVTITADKSTSTAYMELSSLRSED cbets HC 30 (S56 TAVYYCARDGYSLYYFDYWGQGTLVTVLED C) (N69Q) - His1 LKNVFPPEVAVFEPSEAEISHTQKATLVCL ATGFYPDHVELSWWVNGKEVHSGVCTDPQP

LKEQPALQDSRYALSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGR DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LC 12 VYYCTQSHTLRTFGGGTKVEIKPDIQNPDP

AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS KDSDVYITDKCVLDMRSMDFKSNSAVAWSQ

KSDFACANAFQNSIIPEDTFFPSPESS T3 Fab-QVQLVQSGAEVKKPGSSVKVSCKASGFAFT Design_2- DYYIHWVRQAPGQGLEWMGWISPGNVNTKY QQQQ.his2 NENFKGRVTITADKSTSTAYMELSSLRSED VH (CD3) - HCJ2- TAVYYCARDGYSLYYFDYWGQGTLVTVLED HC cbets 31 (S56 LKNVFPPEVAVFEPSEAEISHTQKATLVCL C) (N69Q) - ATGFYPDHVELSWWVNGKEVHSGVCTDPQP His2 LKEQPALQDSRYALSSRLRVSATFWQNPRN

HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRAD

EVAVFEPSEAEISHTQKATLVCLATGFYPD CBeta_1_noC CBeta HVELSWWVNGKEVHSGVSTDPQPLKEQPAL 32 ys (C74A) NDSRYALSSRLRVSATFWQNPRNHFRCQVQ

FYGLSENDEWTQDRAKPVTQIVSAEA

EVAVFEPSEAEISHTQKATLVCLATGFYPD CBeta (S56 HVELSWWVNGKEVHSGVCTDPQPLKEQPAL CBeta_1 33 C) (C74A) NDSRYALSSRLRVSATFWQNPRNHFRCQVQ

FYGLSENDEWTQDRAKPVTQIVSAEA

EVAVFEPSEAEISHTQKATLVCLATGFYPD CBeta (S56 HVELSWWVNGKEVHSGVCTDPQPLKEQPAL CBeta_1-Q 34 C) (N69Q) (C74A)

QDSRYALSSRLRVSATFWQNPRNHFRCQVQ FYGLSENDEWTQDRAKPVTQIVSAEA

EVAVFEPSEAEISHTQKATLVCLATGFYPD CBeta (S56 HVELSWWVNGKEVHSGVCTDPQPLKEQPAL CBeta_1-A 35 C) (N69A) (C74A) ADSRYALSSRLRVSATFWQNPRNHFRCQVQQ

FYGLSENDEWTQDRAKPVTQIVSAEA

EVAVFEPSEAEISHTQKATLVCLATGFYPD CBeta (S56 HVELSWWVNGKEVHSGVCTDPQPLKEQPAL CBeta_1-E 36 C) (N69E) (C74A) EDSRYALSSRLRVSATFWQNPRNHFRCQVQ

FYGLSENDEWTQDRAKPVTQIVSAEA

EVAVFEPSEAEISHTQKATLVCLATGFYPD cbets cbets (S56 HVELSWWVNGKEVHSGVCTDPQPLKEQPAL CBeta_2 37 C) (FG-) (C74A) -------------- NDSRYALSSRLRVSATFWQNPRNHFRCQVQ FYPSN QIVSAEA cbets (S56 EVAVFEPSEAEISHTQKATLVCLATGFYPD C) (N69Q) HVELSWWVNGKEVHSGVCTDPQPLKEQPAL CBeta_2 Q-38 (FG -) QDSRYALSSRLRVSATFWQNPRNHFRCQVQ (C74A) -------------- FYPSN QIVSAEA cbets (S56 EVAVFEPSEAEISHTQKATLVCLATGFYPD C) (N69A) CBeta_2 HVELSWWVNGKEVHSGVCTDPQPLKEQPAL 39-A (FG-) ADSRYALSSRLRVSATFWQNPRNHFRCQVQ (C74A) FYPSN ----- --------- QIVSAEA CBeta (S56 EVAVFEPSEAEISHTQKATLVCLATGFYPD C) (N69E) HVELSWWVNGKEVHSGVCTDPQPLKEQPAL CBeta_2-E 40 (FG-) (C74A)

EDSRYALSSRLRVSATFWQNPRNHFRCQVQ FYPSN -------------- QIVSAEA

EVAVFEPSEAEISHTQKATLVCLATGFYPD CBeta (S56 HVELSWWVNGKEVHSGVCTDPQPLKEQ --- CBeta_3 41 C) (FG-DE-) S- (C74A) GRYALSSRLRVSATFWQNPRNHFRCQVQFEA PSN -------------- QIVSA

AVYQLRDSKSSDKSVCLFTDFDSQTNVSQS CAlpha_1_no Cys 42 CAlpha KDSDVYITDKTVLDMRSMDFKSNSAVAWSN

KSDFACANAFNNSIIPEDTFFPSPESS

AVYQLRDSKSSDKSVCLFTDFDSQTNVSQS CAlpha CAlpha_1 43 (T49C)

KDSDVYITDKCVLDMRSMDFKSNSAVAWSN

KSDFACANAFNNSIIPEDTFFPSPESS CAlpha AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS CAlpha_1- (T49C) KDSDVYITDKCVLDMRSMDFKSNSAVAWSQ 44 QQQ (N34Q + N68 Q + N79Q)

KSDFACANAFQNSIIPEDTFFPSPESS CAlpha AVYQLRDSKSSDKSVCLFTDFDSQTAVSQS CAlpha_1- (T49C) KDSDVYITDKCVLDMRSMDFKSNSAVAWSA 45 AAA (N34A + N68 A + N79A)

KSDFACANAFANSIIPEDTFFPSPESS CAlpha AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS CAlpha_1- (T49C) KDSDVYITDKCVLDMRSMDFKSNSAVAWSS CAlpha 46 QSK (N34Q + N68 S + N79K)

KSDFACANAFKNSIIPEDTFFPSPESS CAlpha AVYQLRDSKSSDKSVCLFTDFDSQTAVSQS CAlpha_1- (T49C) KDSDVYITDKCVLDMRSMDFKSNSAVAWSS 47 ASK (N34A + N68 S + N79K)

KSDFACANAFKNSIIPEDTFFPSPESS CAlpha AVYQLRDSKSSDKSVCLFTDFDSQTQVSQS (T49C) KDSDVYITDKCVLDMRSMDFKSQSAVAWSQ CAlpha_1- (N34Q + N68

QQQQ 48 Q +

N61 Q + N79Q KSDFACANAFQNSIIPEDTFFPSPESS) H_Conjunctio 49 VH SSASKNVFPP n_1 CBetaCJ1 ConjunctionX H_Conjunctio 50 LEDLKNVFPP n_2 VH VL CBetaCJ2 L_Conjunctio 51 KRTVAAPDP n_1 CAlphaCJ1 ConjunctionZ L_Conjunctio 52 VL KPDIQNPDP n_2 CAlphaCJ2 Conjunction'_I CBeta- WGRASDKTHTCPPCPAPEAAGGP Conjunction 53 'gG1 (IgG1) CJ1 Conjunction'Y Conjunction'_ CBeta- WGR --- YGPPCPPCPAPEFLGGP 54 Conjunction '1gG4 (IgG4) CJ1 307 FE 308 VH- KLEDLKNVFPP CBetaCJ2 309 VL-Cpre- KPTGVGGTP AlphaCJB Cter (WGQ chain light name 306 cross-stitch name) Pre-Alpha-Beta Chain No

DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGGT LC 64 CPreAlpha FPSLQPGV

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED Design_1_Pr LKNVFPPEVAVFEPSEAEISHTQKATLVCL e_TCR_Conj ATGFYPDHVELSWWVNGKEVHSGVSTDPQP unction'1 VH (CD3) - LKEQPALQDSRYALSSRLRVSATFWQNPRN HCJB- HFRCQVQFYGLSENDEWTQDRAKPVTQIVS cbets HC 65 (N69 Q) -CJ'1G1- AEAWGRASDKTHTCPPCPAPEAAGGPSVFL Fc (G1) FPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP LC 66 CPreAlpha (Y59CQVGGVTGVLVLGV)

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED Design_2_Pr LKNVFPPEVAVFEPSEAEISHTQKATLVCL e_TCR_Conj ATGFYPDHVELSWWVNGKEVHSGVSTDPQP unction'1_Cys VH (CD3) - 10 HCJB- LKEQPALQDSRYALCSRLRVSATFWQNPRN cbets (S76 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 67 C) (N69Q) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK VL (CD3) -

LC DIVMTQSPDSLAVSLGERATINCKSSQSLL LCJB- NSRTRKNYLAWYQQKPGQPPKLLIYWASTR 68 CPreAlpha QSGVPDRFSGSGSGTDFTLTISSLQAEDVA (A13C) VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP Design_3_Pr (N50Q) FPSLCPPIMLLVDGKQQMVVVCLVLDVAPP e_TCR_Conj unction'1_Cys GLDSPIWFSAGQGSALDAFTYGPSPATDGT 11 WTNLAHLSLPSEELASWEPLVCHTGPGAEG

HSRSTQPMHLSGEASTART QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVLED LKNVFPPEVAVCEPSEAEISHTQKATLVCL

ATGFYPDHVELSWWVNGKEVHSGVSTDPQP VH (CD3) - HCJB- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (F13 HC HFRCQVQFYGLSENDEWTQDRAKPVTQIVS 69 C) (N69Q) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP LC 68 CPreAlpha (A13QQVGGPFT)

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED Design_4_Pr LKNVFPPEVAVFEPCEAEISHTQKATLVCL e_TCR_Conj ATGFYPDHVELSWWVNGKEVHSGVSTDPQP unction'1_Cys VH (CD3) - 12 HCJB- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (S16 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 70 C) (N69Q) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP CPreAlpha LC 71 (S11C) FPCLAPPIMLLVDGKQQMVVVCLVLDVAPP Design_5_Pr (N50Q) GLDSPIWFSAGQGSALDAFTYGPSPATDGT e_TCR_Conj WTNLAHLSLPSEELASWEPLVCHTGPGAEG unction'1_Cys HSRSTQPMHLSGEASTART 13 VH (CD3) - HC 70 QVQLVQSGAEVKKPGSSVKVSCKASGFAFT HCJB- DYYIHWVRQAPGQGLEWMGWISPGNVNTKY cbets (S16

C) (N69Q) - NENFKGRVTITADKSTSTAYMELSSLRSED CJ’1G1- TAVYYCARDGYSLYYFDYWGQGTLVTVLED Fc (G1)

LKNVFPPEVAVFEPCEAEISHTQKATLVCL ATGFYPDHVELSWWVNGKEVHSGVSTDPQP LKEQPALQDSRYALSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRASDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP LC 71 CPreAlpha (S11C)

FPCLAPPIMLLVDGKQQMVVVCLVLDVAPP (N50Q) GLDSPIWFSAGQGSALDAFTYGPSPATDGT

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED Design_6_Pr LKNVFPPEVAVFEPSECEISHTQKATLVCL e_TCR_Conj ATGFYPDHVELSWWVNGKEVHSGVSTDPQP unction'1_Cys VH (CD3) - 14 HCJB- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (A18 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 72 C) (N69Q) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGFTTLDV (G11QPPVGGGTP)

WTNLAHLSLPSEELASWEPLVCHTGPGAEG

HSRSTQPMHLSGEASTART Design_7_Pr e_TCR_Conj QVQLVQSGAEVKKPGSSVKVSCKASGFAFT unction’1_Cys VH (CD3) - DYYIHWVRQAPGQGLEWMGWISPGNVNTKY 15 HCJB- NENFKGRVTNY

TAVYYCARDGYSLYYFDYWGQGTLVTVLED CJ’1G1- LKNVFPPEVAVFEPSEACISHTQKATLVCL Fc (G1) ATGFYPDHVELSWWVNGKEVHSGVSTDPQP

LKEQPALQDSRYALSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRASDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP LC 74 CPreAlpha (S62CQPVVGFT)

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED Design_8_Pr LKNVFPPEVAVFEPSEAEISHTQKATLVCL e_TCR_Conj ATGFYPDHVELSWWVNGKEVHSGVCTDPQP unction'1_Cys VH (CD3) - 1_4L4T_1 HCJB- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (S56 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 75 C) (N69Q) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP LC 76 CPreAlpha (T65QQVVGGVTGVTLVLGTLQQQVLQQVGGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTLGTTTTTTTTTTTTGGTTTTTTTTTTGGTTTTTGTGTGTTGTGTXTGTGTGT either WTF (F65F) F

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART QVQLVQSGAEVKKPGSSVKVSCKASGFAFT

DYYIHWVRQAPGQGLEWMGWISPGNVNTKY Design_9_Pr NENFKGRVTITADKSTSTAYMELSSLRSED e_TCR_Conj TAVYYCARDGYSLYYFDYWGQGTLVTVLED unction'1_Cys VH (CD3) - 2_4L4T_2 HCJB- LKNVFPPEVAVFEPSEAEISHTQKATLVCL cbets (S56 ATGFYPDHVELSWWVNGKEVHSGVCTDPQP HC 75 C) (N69Q) - LKEQPALQDSRYALSSRLRVSATFWQNPRN CJ'1G1- HFRCQVQFYGLSENDEWTQDRAKPVTQIVS Fc (G1)

AEAWGRASDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJB- VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP LC 77 CPreAlpha (I16CQVVGDLVLGGL)

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED Design_10_P LKNVFPPEVCVFEPSEAEISHTQKATLVCL re_TCR_Conj ATGFYPDHVELSWWVNGKEVHSGVSTDPQP unction'1_Cys VH (CD3) - 4 HCJB- LKEQPALQDSRYALSSRLRVSATFWQNPRN cbets (A11 HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 78 C) (N69Q) - AEAWGRASDKTHTCPPCPAPEAAGGPSVFL CJ'1G1- FPPKPKDTLMISRTPEVTCVVVDVSHEDPE Fc (G1)

VKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR QSGVPDRFSGSGSGTDFTLTISSLQAEDVA

VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP LC 58 FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP

GLDSPIWFSAGQGSALDAFTYGPSPATDGT WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTARTCPQEPLRGTPG G QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY

NENFKGRVTITADKSTSTAYMELSSLRSED Design_11_P

TAVYYCARDGYSLYYFDYWGQGTLVTVLED re_TCR_Conj LKNVFPPEVAVFEPSEAEISHTQKATLVCL unction’2_CT ATGFYPDHVELSWWVNGKEVHSGVSTDPQP erminal LKEQPALQDSRYALSSRLRSS

HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 79

AEAWGRADCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR QSGVPDRFSGSGSGTDFTLTISSLQAEDVA

VYYCTQSHTLRTFGGGTKVEIKPTGVGGTP LC 63

FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP GLDSPIWFSAGQGSALDAFTYGPSPATDGT WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTARTC QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVLED Design_12_P LKNVFPPEVAVFEPSEAEISHTQKATLVCL re_TCR_Conj ATGFYPDHVELSWWVNGKEVHSGVSTDPQP unction’3_C LKEQPALQDSRYALSSRLR

HFRCQVQFYGLSENDEWTQDRAKPVTQIVS HC 80

AEAWGRADCGFTSVCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK ConjunctionW L_Conjunctio 81 VL-Cpre- PTGVGGTP n_3 AlphaCJB

FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP

GLDSPIWFSAGNGSALDAFTYGPSPATDGT CPreAlpha 82

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART

FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- GLDSPIWFSAGQGSALDAFTYGPSPATDGT (N50Q) noGlyco 83 (Design1)

WTNLAHLSLPSEELASWEPLVCHTGPGAEG HSRSTQPMHLSGEASTART

FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- GLDSPIWFSAGQGSALDAFTCGPSPATDGT (N50Q, noGlyco-Cys 311 (Design2) Y59C) WTNLAHLSLPSEELASWEPLVCHTGPGAEG

HSRSTQPMHLSGEASTART

FPSLCPPIMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- GLDSPIWFSAGQGSALDAFTYGPSPATDGT (N50Q, noGlyco-Cys 312 (Design3) A13C) WTNLAHLSLPSEELASWEPLVCHTGPGAEG

HSRSTQPMHLSGEASTART CPreAlpha- (N50Q, noGlyco-Cys 312 A13C) (Design4)

FPCLAPPIMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- GLDSPIWFSAGQGSALDAFTYGPSPATDGT (N50Q, noGlyco-Cys 313 (Design5) S11C) WTNLAHLSLPSEELASWEPLVCHTGPGAEG CPreAlpha

HSRSTQPMHLSGEASTART CPreAlpha- (N50Q, noGlyco-Cys 313 S11C) (Design6) CPreAlpha- (N50Q, noGlyco-Cys 313 S11C) (Design7)

FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- GLDSPIWFSAGQGSALDAFTYGPCPATDGT (N50Q, noGlyco-Cys 314 (Design8) S62C) WTNLAHLSLPSEELASWEPLVCHTGPGAEG

HSRSTQPMHLSGEASTART

FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- GLDSPIWFSAGQGSALDAFTYGPSPACDGT (N50Q, noGlyco-Cys 315 (Design9) T65C) WTNLAHLSLPSEELASWEPLVCHTGPGAEG

HSRSTQPMHLSGEASTART

FPSLAPPCMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- GLDSPIWFSAGQGSALDAFTYGPSPATDGT (N50Q, noGlyco-Cys 316 (Design10) I16C) WTNLAHLSLPSEELASWEPLVCHTGPGAEG

HSRSTQPMHLSGEASTART

FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- (N50Q, GLDSPIWFSAGQGSALDAFTYGPSPATDGT noGlyco 317 ter_residues WTNLAHLSLPSEELASWEPLVCHTGPGAEG (Design11)) HSRSTQQGGGE

G

FPSLAPPIMLLVDGKQQMVVVCLVLDVAPP CPreAlpha- (N50Q, GLDSPIWFSAGQGSALDAFTYGPSPATDGT noGlyco 318 Cter_somen (Design12) te) WTNLAHLSLPSEELASWEPLVCHTGPGAEG

HSRSTQPMHLSGEASTARTC

EVAVFEPSEAEISHTQKATLVCLATGFYPD CBeta_for HVELSWWVNGKEVHSGVSTDPQPLKEQPAL 84 N69Q PreAlpha QDSRYALSSRLRVSATFWQNPRNHFRCQVQ

FYGLSENDEWTQDRAKPVTQIVSAEA Design_1 84 N69Q

EVAVFEPSEAEISHTQKATLVCLATGFYPD N69Q, HVELSWWVNGKEVHSGVSTDPQPLKEQPAL Design_2 319 S76C QDSRYALCSRLRVSATFWQNPRNHFRCQVQ

FYGLSENDEWTQDRAKPVTQIVSAEA

EVAVCEPSEAEISHTQKATLVCLATGFYPD N69Q, HVELSWWVNGKEVHSGVSTDPQPLKEQPAL Design_3 320 F13C QDSRYALSSRLRVSATFWQNPRNHFRCQVQ

FYGLSENDEWTQDRAKPVTQIVSAEA

EVAVFEPCEAEISHTQKATLVCLATGFYPD N69Q, HVELSWWVNGKEVHSGVSTDPQPLKEQPAL Design_4 321 S16C QDSRYALSSRLRVSATFWQNPRNHFRCQVQ Cbeta_for_Cp FYGLSENDEWTQDRAK 321V,

EVAVFEPSECEISHTQKATLVCLATGFYPD N69Q, HVELSWWVNGKEVHSGVSTDPQPLKEQPAL Design_6 322 A18C QDSRYALSSRLRVSATFWQNPRNHFRCQVQ

FYGLSENDEWTQDRAKPVTQIVSAEA

EVAVFEPSEACISHTQKATLVCLATGFYPD N69Q, HVELSWWVNGKEVHSGVSTDPQPLKEQPAL Design_7 323 E19C QDSRYALSSRLRVSATFWQNPRNHFRCQVQQ

FYGLSENDEWTQDRAKPVTQIVSAEA N69Q, Design_8 34 S56C

N69Q, Design_9 34 S56C

EVCVFEPSEAEISHTQKATLVCLATGFYPD N69Q, HVELSWWVNGKEVHSGVSTDPQPLKEQPAL Design_10 324 A11C QDSRYALSSRLRVSATFWQNPRNHFRCQVQ

FYGLSENDEWTQDRAKPVTQIVSAEA Design_11 84 N69Q Design_12 84 N69Q Drawings SEQ name ID Sequences Delta-Gamma String NO

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ5- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LC 85 CDelta VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK (Q PSVFVMKQGTNVACLVKEFYPKDIRINLVS N16Q + N79) SKKITEFDPAIVISPSGKYNAVKLGKYEDS

NSVTCSVQHDQKTVHSTDFE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVTDK

QLDADVSPKPTIFLPSIAETKLQKAGTYLC dg_Design_2 LLEKFFPDVIKIHWQEKKSNTILGSQEGNT VH (CD3) - _no_Glyco HCJ5- MKTQDTYMKFSWLTVPEESLDKEHRCIVRH CGamma- ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP HC 86 CJ'3G1- EAAGGPSVFLFPPKPKDTLMISRTPEVTCV Fc (G1) VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR (N65Q)

EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) -

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ5- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LC 89 CDelta VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK (N16C) PSVFVMKKGY

NSVTCSVQHDQKTVHSTDFE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT

DYYIHWVRQAPGQGLEWMGWISPGNVNTKY dg_Design_2 NENFKGRVTITADKSTSTAYMELSSLRSED VH (CD3) - _hypeCys1_n HCJ5- TAVYYCARDGYSLYYFDYWGQGTLVTVTDK o_Glyco Cgamma QLDADVSPKPCIFLPSIAETKLQKAGTYLC HC 90 (T12C) LLEKFFPDVIKIHWQEKKSNTILGSQEGNT (N65Q) - MKTQDTYMKFSWLTVPEESLDKEHRCIVRH CJ'3G1- ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP Fc (G1)

EAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ5- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CDelta VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK LC 91 (V50C) (PSVFVMKQGTNVACLVKEFYPKDIRINLVS N79Q N16Q +) SKKITEFDPAICISPSGKYNAVKLGKYEDS

NSVTCSVQHDQKTVHSTDFE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVTDK

QLDADVSPKPTIFLPSIAETKLQKAGTYLC dg_Design_2 VH (CD3) - LLEKFFPDVIKIHWQEKKSNTILGSCEGNT _hypeCys2_n HCJ5- MKTQDTYMKFSWLTVPEESLDKEHRCIVRH o_Glyco Cgamma ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP HC 92 (Q57C) (N65Q) - EAAGGPSVFLFPPKPKDTLMISRTPEVTCV CJ'3G1- VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR Fc (G1) EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ5- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CDelta LC 93 (D46C)

VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK (N16Q + PSVFVMKQGTNVACLVKEFYPKDIRINLVS N79Q) SKKITEFCPAIVISPSGKYNAVKLGKYEDS

NSVTCSVQHDQKTVHSTDFE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVTDK

QLDADVSPKPTIFLPSIAETKLQKAGTYLC dg_Design_2 VH (CD3) - LLEKFFPDVIKIHWQEKKSNTILGSQEGNT _hypeCys3_n HCJ5- CKTQDTYMKFSWLTVPEESLDKEHRCIVRH o_Glyco Cgamma ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP HC 94 (M62C) (N65Q) - EAAGGPSVFLFPPKPKDTLMISRTPEVTCV CJ'3G1- VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR Fc (G1) EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS

LSPGK VL (CD3) - DIVMTQSPDSLAVSLGERATINCKSSQSLL LC 95 LCJ5- NSRTRKNYLAWYQQKPGQPPKLLIYWASTR CDelta

(F12C) QSGVPDRFSGSGSGTDFTLTISSLQAEDVA dg_Design_2 (N16Q + VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK _Cys2_no_Gl N79Q) yco

PSVCVMKQGTNVACLVKEFYPKDIRINLVS SKKITEFDPAIVISPSGKYNAVKLGKYEDS NSVTCSVQHDQKTVHSTDFE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVTDK

QLDADVSPKPTIFLPCIAETKLQKAGTYLC VH (CD3) - LLEKFFPDVIKIHWQEKKSNTILGSQEGNT HCJ5- MKTQDTYMKFSWLTVPEESLDKEHRCIVRH Cgamma ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP HC 96 (S17C) (N65Q) - EAAGGPSVFLFPPKPKDTLMISRTPEVTCV CJ'3G1- VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR Fc (G1) EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ5- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CDelta VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK LC 97 (M14C) (PSVFVCKQGTNVACLVKEFYPKDIRINLVS N79Q N16Q +) SKKITEFDPAIVISPSGKYNAVKLGKYEDS

NSVTCSVQHDQKTVHSTDFE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVTDK

QLDADVSPKPTICLPSIAETKLQKAGTYLC dg_Design_2 VH (CD3) - LLEKFFPDVIKIHWQEKKSNTILGSQEGNT _Cys1_no_Gl HCJ5- MKTQDTYMKFSWLTVPEESLDKEHRCIVRH YCO Cgamma ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP HC 98 (F14C) (N65Q) - EAAGGPSVFLFPPKPKDTLMISRTPEVTCV CJ'3G1- VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR Fc (G1) EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ5- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CDelta LC 95 (F12C)

VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK (PSVCVMKQGTNVACLVKEFYPKDIRINLVS N16Q + N79Q dg_Design_2) SKKITEFDPAIVISPSGKYNAVKLGKYEDS _Cys3_no_Gl NSVTCSVQHDQKTVHSTDFE YCO VH (CD3) - HC 99 QVQLVQSGAEVKKPGSSVKVSCKASGFAFT HCJ5- DYYIHWVRQAPGQGLEWMGWISPGNVNTKY Cgamma

(E20C) NENFKGRVTITADKSTSTAYMELSSLRSED (N65Q) - TAVYYCARDGYSLYYFDYWGQGTLVTVTDK CJ’3G1- Fc (G1)

QLDADVSPKPTIFLPSIACTKLQKAGTYLC LLEKFFPDVIKIHWQEKKSNTILGSQEGNT MKTQDTYMKFSWLTVPEESLDKEHRCIVRH ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ5- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CDelta VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK LC 100 (F87C) (PSVFVMKQGTNVACLVKEFYPKDIRINLVS N79Q N16Q +) SKKITEFDPAIVISPSGKYNAVKLGKYEDS

NSVTCSVQHDQKTVHSTDCE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVTDK

QLDADVSPKPTIFLPSICETKLQKAGTYLC dg_Design_2 VH (CD3) - LLEKFFPDVIKIHWQEKKSNTILGSQEGNT _Cys4_no_Gl HCJ5- MKTQDTYMKFSWLTVPEESLDKEHRCIVRH YCO Cgamma ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP 101 HC (A19C) (N65Q) - EAAGGPSVFLFPPKPKDTLMISRTPEVTCV CJ'3G1- VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR Fc (G1) EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK

VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL (CD3) - NSRTRKNYLAWYQQKPGQPPKLLIYWASTR LCJ5- QSGVPDRFSGSGSGTDFTLTISSLQAEDVA CDelta LC 102 (E88C)

VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK (N16Q + PSVFVMKQGTNVACLVKEFYPKDIRINLVS N79Q) SKKITEFDPAIVISPSGKYNAVKLGKYEDS

NSVTCSVQHDQKTVHSTDFC

QVQLVQSGAEVKKPGSSVKVSCKASGFAFT dg_Design_2 DYYIHWVRQAPGQGLEWMGWISPGNVNTKY VH (CD3) - _Cys5_no_Gl HCJ5-

NENFKGRVTITADKSTSTAYMELSSLRSED yco CGamma TAVYYCARDGYSLYYFDYWGQGTLVTVTDK HC 101 (A19C) QLDADVSPKPTIFLPSICETKLQKAGTYLC (N65Q) - LLEKFFPDVIKIHWQKKTHKTKKTKHKTKKTKY

ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LC 105 LCJ5- VYYCTQSHTLRTFGGGTKVEIEPRSQPHTK CDelta PSVFVMKNGT

SKKITEFDPAIVISPSGKYNAVKLGKYEDS NSVTCSVQHDNKTVHSTDFE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVTDK QLDADVSPKPTIFLPSIAETKLQKAGTYLC

LLEKFFPDVIKIHWQEKKSNTILGSQEGNT dg_Design_2 VH (CD3) - MKTNDTYMKFSWLTVPEESLDKEHRCIVRH HCJ5- ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP HC 106 CGamma- CJ'3G1- EAAGGPSVFLFPPKPKDTLMISRTPEVTCV Fc (G1) VVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LC 107 LCJ4- VYYCTQSHTLRTFGGGTKVEIKPRSQPHTK CDelta PSVFVMKNGT

SKKITEFDPAIVISPSGKYNAVKLGKYEDS NSVTCSVQHDNKTVHSTDFE QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED TAVYYCARDGYSLYYFDYWGQGTLVTVSSA SLDADVSPKPTIFLPSIAETKLQKAGTYLC

LLEKFFPDVIKIHWQEKKSNTILGSQEGNT dg_Design_1 VH (CD3) - MKTNDTYMKFSWLTVPEESLDKEHRCIVRH HCJ4- ENNKNGVDQEIIFPPIKSDKTHTCPPCPAP HC 108 CGamma- CJ '

EEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA VL (CD3) - VYYCTQSHTLRTFGGGTKVEIKDKQLDADV LC 109 LCJ6- CGamma SPKPTIFLPSIAETKLQKAGTYLCLLEKFF

PDVIKIHWQEKKSNTILGSCEGNTMKTQDT YMKFSWLTVPEESLDKEHRCIVRHENNKNG VDQEIIFPPIKTDVITMD QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVSSR dg_crossed_ SQPHTKPSVFVMKQGTNVACLVKEFYPKDI Design_1 VH (CD3) -

RINLVSSKKITEFDPAICISPSGKYNAVKL HCJ6- GKYEDSNSVTCSVQHDQKTVHSTDEPKSCD HC 110 CDelta- KTHTCPPCPAPEAAGGPSVFLFPPKPKDTL CJ’4G1- MISRTPEVTCVVVDVSHVTVNVHVNQVHVHVVVVVVVVVVHVVVVVVHVNQQQQNVNQVVVVVVVVVVVVVVVVTKKVNKHHHHHHHHH

DWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL NSRTRKNYLAWYQQKPGQPPKLLIYWASTR

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA VL (CD3) - VYYCTQSHTLRTFGGGTKVEIKDKQLDADV LC 111 LCJ7- CGamma SPKPTIFLPSIAETKLQKAGTYLCLLEKFF

PDVIKIHWQEKKSNTILGSCEGNTMKTQDT YMKFSWLTVPEESLDKEHRCIVRHENNKNG VDQEIIFPPIKTDVITMD QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVEPR dg_crossed_ SQPHTKPSVFVMKQGTNVACLVKEFYPKDI Design_2 VH (CD3) -

RINLVSSKKITEFDPAICISPSGKYNAVKL HCJ7- GKYEDSNSVTCSVQHDQKTVHSTDEPKSCD HC 112 CDelta- KTHTCPPCPAPEAAGGPSVFLFPPKPKDTL CJ’4G1- MISRTPEVTCVVVDVSHTLVNVHVNQVVVVVVVVVVVVVHVHQHVNQQVVVVVVVVVVVVVVVVTKKVNKKNYVNKVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVKVKHKHHHHHHHHHHHH

DWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK KPTIFLPSIAETKLQKAGTYLCLLEKFFPD

VIKIHWQEKKSNTILGSQEGNTMKTNDTYM CGamma_1 113 CGamma

KFSWLTVPEESLDKEHRCIVRHENNKNGVD CGamma

QEIIF CGamma_1_ CGamma KPTIFLPSIAETKLQKAGTYLCLLEKFFPD 114 no_Glyco (N65Q) VIKIHWQEKKSNTILGSQEGNTMKTQDTYM

KFSWLTVPEESLDKEHRCIVRHENNKNGVD

QEIIF dg_Design_1 Without 113 mutations Without dg_Design_2 113 mutations dg_Design_2 114 N65Q _no_Glyco

KPCIFLPSIAETKLQKAGTYLCLLEKFFPD dg_Design_2 VIKIHWQEKKSNTILGSQEGNTMKTQDTYM N65Q, _hypeCys1_n 333 o_Glyco T12C KFSWLTVPEESLDKEHRCIVRHENNKNGVD

QEIIF

KPTIFLPSIAETKLQKAGTYLCLLEKFFPD dg_Design_2 VIKIHWQEKKSNTILGSCEGNTMKTQDTYM N65Q, _hypeCys2_n 334 o_Glyco Q57C KFSWLTVPEESLDKEHRCIVRHENNKNGVD

QEIIF

KPTIFLPSIAETKLQKAGTYLCLLEKFFPD dg_Design_2 VIKIHWQEKKSNTILGSQEGNTCKTQDTYM N65Q, _hypeCys3_n 335 o_Glyco M62C KFSWLTVPEESLDKEHRCIVRHENNKNGVD

QEIIF

KPTIFLPCIAETKLQKAGTYLCLLEKFFPD dg_Design_2 VIKIHWQEKKSNTILGSQEGNTMKTQDTYM N65Q, _Cys2_no_Gl 336 yco S17C KFSWLTVPEESLD-

KEHRCIVRHENNKNGVDQEIIF

KPTICLPSIAETKLQKAGTYLCLLEKFFPD dg_Design_2 VIKIHWQEKKSNTILGSQEGNTMKTQDTYM N65Q, _Cys1_no_Gl 337 yco F14C KFSWLTVPEESLDKEHRCIVRHENNKNGVDD

QEIIF

KPTIFLPSIACTKLQKAGTYLCLLEKFFPD dg_Design_2 VIKIHWQEKKSNTILGSQEGNTMKTQDTYM N65Q, _Cys3_no_Gl 338 yco E20C KFSWLTVPEESLDKEHRCIVRHENNKNGVDD

QEIIF

KPTIFLPSICETKLQKAGTYLCLLEKFFPD dg_Design_2 VIKIHWQEKKSNTILGSQEGNTMKTQDTYM N65Q, _Cys4_no_Gl 339 yco A19C KFSWLTVPEESLDKEHRCIVRHENNKNGVD

QEIIF dg_Design_2 N65Q, _Cys5_no_Gl 339 A19C yco

KPTIFLPSIAETKLQKAGTYLCLLEKFFPD dg_crossed_ VIKIHWQEKKSNTILGSCEGNTMKTQDTYM 340 Design_1 KFSWLTVPEESLDKEHRCIVRHENNKNGVD

QEIIFPPIKTDVITMD dg_crossed_ 340 Design_2

SVFVMKNGTNVACLVKEFYPKDIRINLVSS CDelta_1 115 CDelta KKITEFDPAIVISPSGKYNAVKLGKYEDSN

SVTCSVQHDNKTVHSTDFE

SVFVMKQGTNVACLVKEFYPKDIRINLVSS CDelta_1_no CDelta (N16 CDelta _Glyco 116 Q + N79Q)

KKITEFDPAIVISPSGKYNAVKLGKYEDSN SVTCSVQHDQKTVHSTDFE

KSVFVMKNGTNVACLVKEFYPKDIRINLVS 310 CDelta SKKITEFDPAIVISPSGKYNAVKLGKYEDS

NSVTCSVQHDNKTVHSTDFE dg_Design_1 No 115 mutations No dg_Design_2 115 mutations dg_Design_2 N16Q, 116 _no_Glyco N79Q dg_Design_2 SVFVMKCGTNVACLVKEFYPKDIRINLVSS N16C, _hypeCys1_n 325 KKITEFDPAIVISPSGKYNAVKLGKYEDSN N79Q o_Glyco SVTCSVQHDQKTVHSTDFE dg_Design_2 N16Q, SVFVMKQGTNVACLVKEFYPKDIRINLVSS _hypeCys2_n 326 N79Q, KKITEFDPAICISPSGKYNAVKLGKYEDSN o_Glyco V50C SVTCSVQHDQKTVHSTDFE dg_Design_2 N16Q, SVFVMKQGTNVACLVKEFYPKDIRINLVSS _hypeCys3_n 327 N79Q, KKITEFCPAIVISPSGKYNAVKLGKYEDSN o_Glyco D46C SVTCSVQHDQKTVHSTDFE dg_Design_2 N16Q, N79Q SVCVMKQGTNVACLVKEFYPKDIRINLVSS _Cys2_no_Gl 328, YCO KKITEFDPAIVISPSGKYNAVKLGKYEDSN F12C SVTCSVQHDQKTVHSTDFE dg_Design_2 N16Q, N79Q SVFVCKQGTNVACLVKEFYPKDIRINLVSS _Cys1_no_Gl 329, YCO KKITEFDPAIVISPSGKYNAVKLGKYEDSN M14C SVTCSVQHDQKTVHSTDFE dg_Design_2 N16Q, N79Q _Cys3_no_Gl 328, YCO F12C dg_Design_2 N16Q, N79Q SVFVMKQGTNVACLVKEFYPKDIRINLVSS _Cys4_no_Gl 330, YCO KKITEFDPAIVISPSGKYNAVKLGKYEDSN F87C SVTCSVQHDQKTVHSTDCE dg_Design_2 N16Q, SVFVMKQGTNVACLVKEFYPKDIRI N79Q NLVSS _Cys5_no_Gl 331, YCO KKITEFDPAIVISPSGKYNAVKLGKYEDSN E88C SVTCSVQHDQKTVHSTDFC dg_crossed_ SVFVMKQGTNVACLVKEFYPKDIRINLVSS design_1 N16Q, KKITEFDPAICISPSGKYNAVKLGKYEDSN (delta 332 in N79Q, V50C SVTCSVQHDQKTVHSTD-- heavy chain) dg_crossed_ Design_2 (delta 332 in the heavy chain) H_Conjunctio 117 HCJ4

SSASLDADVSP n_4 ConjunctionR H_Conjunctio 118 HCJ5 TDKQLDADVSP n_5 L_Conjunctio 119 LCJ4 PRSQPHTKP n_4 ConjunctionT L_Conjunctio 120 LCJ5

EPRSQPHTKP n_5 Conjunction’3 121 CJ’3G1 PPIKSDKTHTCPPCPAPEAAGGP _IgG1 Conjunction’S Conjunction’3 122 CJ’3G4 PPI --- YGPPCPPCPAPEFLGGP _1gG4 H_Conjunctio 123 HCJ6 SSRSQPHTKP_HJ6Conjunc

L_Conjunctio 126 LCJ7 TDKLDADVSP n_7 Conjunction'4 127 CJ'4G1 EPKSSDKTHTCPPCPAPEAAGGP _IgG1 Conjunction'I Conjunction'4 128 CJ'4G4 ESK --- YGPPCPPCPAPEFLGGP _1gG4 Conjunction H_ConjuncH_3JJJ3J3HJ3J3HJ3J3J3 Conjunction H_Conjunctio 130 HCJ4 SSPDIQNPDP (Alpha-Beta, crossed n_4, plus L_Conjunctio 50 LCJ3 TCR) n_3 Conjunction RTVAAGTP (Pre-Alpha- Beta Junction, normal, 131 LCJA light chain plus antibody) Conjunction Junction of 50 LCJC (Pre-Alpha- Beta light chain, cross, plus Junction of SSASGVGGTP chain 132 HCJC antibody) Conjunction heavy Junction of 50 LCJD (Pre-Alpha- light chain Beta, cross, plus Junction of SSPTGVGGTP chain 133 HCJD TCR) heavy CJ'2G1 SDKTHTCPPCPAPEAAGGP Conjunction 'Conjunction' 134 (Alpha-Beta, Pre-Alpha-Beta, CJ'2G4 --YGPPCPPCPAPEFLGGP cross) Conjunction '135

DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) - Take QSGVPDRFSGSGSGTDFTLTISSLQAEDVA HCJB- VYYCTQSHTLRTFGGGTKVEIKLEDLKNVF cbets PPEVAVFEPCEAEISHTQKATLVCLATGFY 136 (N69Q, S16C) - PDHVELSWWVNGKEVHSGVSTDPQPLKEQP CJ'1G ALQDSRYALSSRLRVSATFWQNPRNHFRCQ

VQFYGLSENDEWTQDRAKPVTQIVSAEAWG FROG

QVQLVQSGAEVKKPGSSVKVSCKASGFAFT PreTCR_Desi DYYIHWVRQAPGQGLEWMGWISPGNVNTKY gn5_crossed_ 1 NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVSSA VH (CD3) - SGVGGTPFPCLAPPIMLLVDGKQQMVVVCL HCJC- VLDVAPPGLDSPIWFSAGQGSALDAFTYGP Heavy CPreAlpha (Junction plus 137 (S11C,

SPATDGTWTNLAHLSLPSEELASWEPLVCH antibody) TGPGAEGHSRSTQPMHLSGEASTARTSDKT N50Q) - CJ’2G1- Fc HTCPPCPAPEAAGGPSVFLFPPKPKDTLMI

SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSLSLSPGK VL (CD3) - HCJB- CBeta Leve 136 (N69Q, S16C) - CJ’1G

QVQLVQSGAEVKKPGSSVKVSCKASGFAFT DYYIHWVRQAPGQGLEWMGWISPGNVNTKY NENFKGRVTITADKSTSTAYMELSSLRSED

TAVYYCARDGYSLYYFDYWGQGTLVTVSSP PreTCR_Desi TGVGGTPFPCLAPPIMLLVDGKQQMVVVCL gn_5_crossed VH (CD3) -

VLDVAPPGLDSPIWFSAGQGSALDAFTYGP _2 SPATDGTWTNLAHLSLPSEELASWEPLVCH HCJD- Heavy TGPGAEGHSRSTQPMHLSGEASTARTSDKT CPreAlpha (Junction plus 138 Pre-TCR) (S11) - HTCPPCPAPEAAGGTFPKVTFKV

VHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLL

NSRTRKNYLAWYQQKPGQPPKLLIYWASTR VL (CD3) -

QSGVPDRFSGSGSGTDFTLTISSLQAEDVA LCJC- VYYCTQSHTLRTFGGGTKVEIKLEDLKNVF Light 139 CBeta PPEVAVFEPSECEISHTQKATLVCLATGFY PreTCR_Desi (A18C, PDHKQQFRHQRHQRHQRHQRHRHRQRHRQRHRHRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRWRRRRRRRRRRRRRRRRWRR

VQFYGLSENDEWTQDRAKPVTQIVSAEAWG

RA Heavy (Junction plus 137 antibody) Light 139 PreTCR_Desi gn_6_crossed Heavy _2 (Junction plus 138 TCR) Conjunction 'SDKTHTCPPCPAPEAAGGP (alpha-beta, CJ'2G1 IgG1 140 cross, IgG1) Conjunction' YGPPCPPCPAPEFLGG4 (alpha-beta, 141 cross, IgG4)

QVQLVQSGAEVKKPGSSVKVSCKASGFAFT VH of DYYIHWVRQAPGQGLEWMGWISPGNVNTKY Antibody 300 NENFKGRVTITADKSTSTAYMELSSLRSED Anti-CD3

TAVYYCARDGYSLYYFDYWGQGTLVTV

DIVMTQSPDSLAVSLGERATINCKSSQSLL VL of NSRTRKNYLAWYQQKPGQPPKLLIYWASTR Antibody 301 QSGVPDRFSGSGSGTDFTLTISSLQAEDVA Anti-CD3

VYYCTQSHTLRTFGGGTKVEI SVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

PAPIEKTISKAKGQPREPQVYTLPPSREEM Fc (G1) 302

TKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK SVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGL

PSSIEKTISKAKGQPREPQVYTLPPSQEEM Fc (G4) 303

TKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK

EXAMPLES EXAMPLE 1: Design and construction of chimeric antibody proteins and

TCR

[0443] TCR sequences

[0444] TCRs are heterodimeric proteins composed of two chains.

About 95% of human T cells have TCRs made up of alpha and beta chains, while the remaining 5% have TCRs made up of gamma and delta chains. The human alpha chain constant region has only one TRAC gene. The constant region of the human beta chain has two subclasses: gene TRBC1 and TRBC2.

In the Protein Database (PDB), the number of TRBC1 crystal structures is relatively greater than that of TRBC2; therefore, the TRBC1 sequences were chosen as the main backbone for designing the polypeptide complex disclosed here (“WuXiBody”). A typical amino acid sequence of TRBC1 can be found in the PDL 4L4T structure.

[0445] TCR interchain disulfide bond

[0446] The crystal structures of the TCR were used to guide the construction of WuXiBody. Unlike native TCR anchored to the T cell surface membrane, soluble TCR molecules are less stable, although their 3D structure is very similar to the antibody's Fab. In fact, the instability of the TCR in soluble conditions used to be a major obstacle that prevents the elucidation of its crystalline structure (Wang 2014, supra). A strategy of introducing a pair of Cys mutations into the TCR constant region was adopted and it was found that one can significantly improve the assembly of the chain and improve expression.

[0447] Effects of the disulfide bond on the expression of antibodies

[0448] To determine whether disulfide bonds play a role in maintaining WuXiBody structures, constructions with and / or without disulfide bonds in the TCR constant region of the chimeric antibodies were expressed. The SDS-PAGE results of the expressed WuXiBody were shown in Figures 15-

17. All WuXiBody expressed were complete IgG constructions with two identical arms. Expression of constructions with and without cysteine mutations between S56 in CBeta and T49 in CAlfa, expression of constructions with cysteine mutations between S16 or A18 in CBeta and S11 in CPré-Alpha, between Q57 in CGama and V50 in CDelta and between A19 in CGama and E88 at CDelta was tested.

[0449] The result of the expression of the constructs with the disulfide bond absent in CBeta / CAlfa (SEQ ID NOs: 32/42) indicates that the constructions without disulfide bonds were not able to maintain the antibody structure (see Figure 15B). The expression of the constructs with disulfide bond absent in CBeta / CPré-Alpha and CGama / CDelta was also tested and showed similar results. In contrast, constructs containing mutated cysteine residues were able to form interchain disulfide bonds, which were able to maintain Ig-like structures (see Figure 15A).

Chimeric domain design from WuXiBody

[0450] Cysteine pair mutations (sequence reference numbers in Figures 19A-19E) in the TCR constant regions have been incorporated into different construction drawings for the chimeric antibodies with TCR, which are shown in Table 21.

TABLE 21. Cys mutations paired to introduce interchanged disulfide bond Alpha-Beta Pre-Alpha-Beta Delta-Gamma Protein Name Protein Name Corresponding Correspondent Mutation Mutation Cys pair Cys pair Cys pair SEQ ID NOs. in HC / LC SEQ ID NOs. at HC / LC Design_5_Pre_TCR_Conjun S11-S16 Design_2_Cys2_no_Glyco Y11-S16 ction'1_Cys13 F12-S17 SEQ ID NOs: 96/95 SEQ ID NOs: 70/71 Design_6_Pre_TCR_Conjun Design_2_Cys3_no_Glyco11 99/95 SEQ ID NOs: 72/71 Design_7_Pre_TCR_Conjun Design_2_Cys1_no_Glyco L13-S16 S11-E19 ction'1_Cys15 M14-F14 SEQ ID NOs: 98/97 SEQ ID NOs: 73/71 Design_3_Pre_TCR_Conjun Design_2_h_11_13_16_13 -T12 yco SEQ ID NOs: 69/68 SEQ ID NOs: 90/89 Design_4_Pre_TCR_Conjun Design_2_hypeCys3_no_Gl S16-E14 A13-S16 ction'1_Cys12 D46-M62 yco SEQ ID NOs: 70/68 SEQ ID NOs: 94/93 Design_h_Pre_ Q57 V23-F13 I16-A11 nction'1_Cys4 yco SEQ ID NOs: 78/77 SEQ ID NOs: 92/91 Design_8_Pre_TCR_Conjun Design_2_Cys4_no_Glyco Y44-L62 S62-S56 ction'1_Cys1_4L4T_1 F87-A19 SEQ ID: 101 75/74 Design_9_Pre_TCR_Conjun E88-A19 Design_2_Cys5_no_Glyco T46-D58 T65-S56 ction'1_Cys2_4L4T_2 SEQ SEQ ID NOs: 101/102 ID NOs: 75/76 Design_2_Pre_TCR_Conjun T46-S76 Y59-S76 ction’1_Cys10 SEQ ID NOs: 67/66 T49-S56

Alpha-Beta Pre-Alpha-Beta Delta-Gamma Protein Name Protein Name Corresponding Mutation Mutation Mutations Corresponding Cys pair Cys pair Cys pair SEQ ID NOs. in HC / LC SEQ ID NOs. at HC / LC L51-S56 S62-S56 S62-R78

[0451] For Cys mutations paired in the TCR Alpha-Beta regions, the T49C-S56C disulfide bond was used for all designs.

[0452] The junctions that connect the antibody variable and TCR constant domains, their relative fusion orientations, as well as the Fc connection junctions have been carefully rotated to create a stable and functional WuXiBody. As the structure of the TCR is very similar to the antibody Fab, we overlapped the Fv homology model of the antibody in the variable region of the TCR (PDB 4L4T, Figure 2B). The overlapping structure indicates that the antibody's Fv is structurally compatible with the TCR constant domain. Based on this structural alignment and the corresponding sequences, all relevant construction / modification parameters have been developed, as shown below.

Domain guidance

[0453] As the VH-CBeta / VL-CAlfa and VH-CAlfa / VL-CBeta fusion guidelines could correctly assemble the chimeric protein, we constructed and tested the two orientations. The sequence homology of the VH-VL is closest to the TCR VBeta-VAlfa. We call the formulas VH CBeta / VL-CAlfa as “normal orientation”, and VH-CAlfa / VL-CBeta as “cross orientation”.

First and second join domains

[0454] The antibody and TCR sequences were aligned based on the alignment of the structure and we found that the junctions defined in the germline sequence are not always consistent with what is displayed in the structure.

For example, from the sequence definition, the junctions that connect VH and CH1 should start right after the last two “SS” residues in the VH region. However,

structurally, these two residues are already part of the junction. We define our joins based on structure, not sequence.

[0455] Table 1 and Table 2 in the present disclosure showed the structure-based sequence alignment for two studied orientations.

As it was a challenge to predict which domain would be compatible with which junction domain, we looked at how the antibody and TCR junctions overlapped in the overlapping structures and estimated the possible substitution using one and the other. The drawings of the junction domains were listed in Table 1 and Table 2.

Third join domains

[0456] A strategy similar to that described above was used to align the hinge of human IgG1 and IgG4 with the proximal region of the TCR membrane (ie, the TCR hinge), and their overlap at the structural level was also verified. Table 7 and Table 8 in the present disclosure list the designs of the third junction domains.

FG handle and DE handle

[0457] The alignment of the structures of the constant region of the TCR with that of the antibody revealed that the FG and DE loops of the beta chain of the TCR are longer than the corresponding region in the CH1 of the antibody. Figures 3A-3B show the differences in the constant regions of the beta T cell chain and the heavy chain of the antibody. To test how these two loops could disrupt the structure if CH1 were replaced by TCR beta, constructions with and without these two loops were developed.

[0458] In view of the considerations mentioned above, a total of nine constructions were developed combining these parameters, as listed in Table 22 and Table 23.

TABLE 22. Design of the chimeric proteins (CBeta / CAlfa) of WuXiBody

SEQ ID NOs: (Heavy Chain Orientation Conjunction Conjunction 'Handle FG Handle DE (HC) / Light Chain (LC)) - IgG1 Design_1 Conjunction'_1 Normal Conjunction_1 Native Native SEQ ID NO: 2/1 (IgG1, IgG4) Design_2 Conjunction' _1 Normal Conjunction_2 Native Native SEQ ID NO: 4/3 (IgG1, IgG4) Design_3 Conjunction'_2 Cross Conjunction_3 Native Native SEQ ID NO: 9/8 (IgG1, IgG4) Design_4 Conjunction'_2 Cross Conjunction_4 Native Native SEQ ID NO: 10 / 8 (IgG1, IgG4) Design_5 Conjunction'_1 Normal Conjunction_1 Substituted Native SEQ ID NO: 5/1 (IgG1, IgG4) Design_6 Conjunction'_1 Normal Conjunction_2 Substituted Native SEQ ID NO: 6/3 (IgG1, IgG4) Design_6a Conjunction ' _1 Normal Conjunction_2 Replaced Substituted SEQ ID NO: 7/3 (IgG1, IgG4) Design_7 Conjunction'_2 Crossed Conjunction_3 Native Substituted SEQ ID NO: 9/11 (IgG1, IgG4) Design_8 Conjunction'_2 Crossed Conjunction_4 Substituted Native SEQ ID NO: 10 / 11 (IgG1, IgG4)

TABLE 23. Chimeric protein components and sequences

(CBeta / CAlfa) of WuXiBody Domains from the N-terminal to the C-terminal end and their SEQ ID NOs.

Domain Name First or Third Domain complex and variable Second domain domain of SEQ ID NOs. heavy chain constant of Dimerization junction domain + of the chain: of the TCR antibody (C1 or C2) (D) hinge junction (CJ ') (VH or VL) Design_1 HC VH (CD3) HCJ1 CBeta (S56C) CJ' 1G1 FcG1 SEQ ID NO: 2 SEQ ID NO: 300 SEQ ID NO: 49 SEQ ID NO: 33 SEQ ID NO: 53 SEQ ID NO: 302 Design_1 LC VL (CD3) LCJ1 CAlpha (T49C) SEQ ID NO: 1 SEQ ID NO: 301 SEQ ID NO: 51 SEQ ID NO: 43 Design_2 HC VH (CD3) HCJ2 CBeta (S56C) CJ'1G1 FcG1 SEQ ID NO: 4 SEQ ID NO: 300 SEQ ID NO: 50 SEQ ID NO: 33 SEQ ID NO: 53 SEQ ID NO: 302 Design_2 LC VL (CD3) LCJ2 CAlpha (T49C) SEQ ID NO: 3 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 43 Design_3 HC VH (CD3) HCJ3 CAlpha (T49C) CJ'2G1 FcG1 SEQ ID NO: 9 SEQ ID NO: 300 SEQ ID NO: 129 SEQ ID NO: 43 SEQ ID NO: 134 SEQ ID NO: 302 Design_3 LC VL (CD3) LCJ3 CBeta (S56C) SEQ ID NO: 33 + SEQ ID NO: 8 SEQ ID NO: 301 SEQ ID NO: 308 NO: 306 Design_4 HC VH (CD3) HCJ4 CAlpha (T49C) CJ'2G1 FcG1 SEQ ID NO: SEQ ID NO: 300 SEQ ID NO: 130 SEQ ID NO: 43 SEQ ID NO: 134 SEQ ID NO: 302 10 Design_4 LC VL (CD3) LCJ3 CBeta (S56C) SE Q ID NO: 33 + SEQ ID NO: 8 SEQ ID NO: 301 SEQ ID NO: 308 NO: 306 CBeta (S56C) Design_5 HC VH (CD3) HCJ1 CJ'1G1 FcG1 (FG-) SEQ ID NO: 5 SEQ ID NO: 300 SEQ ID NO: 49 SEQ ID NO: 37 SEQ ID NO: 53 SEQ ID NO: 302 Design_5 LC VL (CD3) LCJ1 CAlpha (T49C) SEQ ID NO: 1 SEQ ID NO: 301 SEQ ID NO: 51 SEQ ID NO: 43

Domains from the N-terminus to the C-terminus and their SEQ ID NOs. Domain Name First or Third Domain complex and variable Second domain domain of SEQ ID NOs. heavy chain constant of Dimerization junction domain + of the chain: of the TCR antibody (C1 or C2) (D) hinge junction (CJ ') (VH or VL) CBeta Design_6 HC VH (CD3) HCJ1 CJ'1G1 FcG1 ( S56C) (FG-) SEQ ID NO: 6 SEQ ID NO: 300 SEQ ID NO: 50 SEQ ID NO: 37 SEQ ID NO: 53 SEQ ID NO: 302 Design_6 LC VL (CD3) LCJ2 CAlpha (T49C) SEQ ID NO : 3 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 43 CBeta Design_6a HC VH (CD3) HCJ2 CJ'1G1 FcG1 (S56C) (DE-FG-) SEQ ID NO: 7 SEQ ID NO: 300 SEQ ID NO: 50 SEQ ID NO: 41 SEQ ID NO: 53 SEQ ID NO: 302 Design_6a LC VL (CD3) LCJ2 CAlpha (T49C) SEQ ID NO: 3 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 43 Design_7 HC VH (CD3) HCJ3 CAlpha (T49C) CJ'2G1 FcG1 SEQ ID NO: 9 SEQ ID NO: 300 SEQ ID NO: 43 SEQ ID NO: 134 SEQ ID NO: 302 SEQ ID NO: 302 CBeta Design_7 LC VL (CD3 ) LCJ3 (S56C) (FG-) SEQ ID NO: SEQ ID NO: 37 + SEQ ID NO: 301 SEQ ID NO: 308 11 NO: 306 Design_8 HC VH (CD3) HCJ4 CAlpha (T49C) CJ'2G1 FcG1 SEQ ID NO: SEQ ID NO: 300 SEQ ID NO: 130 SEQ ID NO: 43 SEQ ID NO: 134 SEQ ID NO: 302 10 CBeta Design_8 LC V L (CD3) LCJ3 (S56C) (FG-) SEQ ID NO: SEQ ID NO: 37 + SEQ ID NO: 301 SEQ ID NO: 308 11 NO: 306 EXAMPLE 2: Generation and characterization of monospecific TCR / antibody chimeras

[0459] Before fusing the TCR domains into bispecific antibody constructs, the feasibility of introducing them into a regular monospecific IgG was evaluated first. An internally developed anti-CD3 antibody, called T3, was selected to conduct this Concept Validation study. The T3 IgG CH1 and CL constant domains were replaced by the corresponding TCR constant region (CAlfa and CBeta). All nine different strategies listed in Table 22 (see above) were applied and all constructions were expressed in the Expi293 system.

[0460] Table 24 listed the expression level of the modified proteins in the supernatants collected and quantified by Q-ELISA. In general, most "normal orientation" designs were better expressed than "cross-orientation" formats, and most "TCR junctions" are better than "antibody junctions". Two “normal orientation” constructions, Design_5 and Design-6 had an expression comparable to that of Design_2). As for the two “cross-oriented” constructions, Design_7, 8 showed a better expression than Design_3,

4. The low expression of the extra-long FG loop in the TCR CBeta was observed, suggesting that this FG loop can cause significant steric clashes with the fused antibody VL domain.

TABLE 24. The expression levels and CD3 binding of all designs in the supernatant Expression Level Concentration (nM) in FACS Samples (IgG1) in Supernatant 5.0 0.4 0.032 (µg / mL) MFI T3 N / A 5101 2937 408 Design_1 72.04 5441 2190 292 Design_2 204.42 5833 2616 380 Design_3 15.35 5089 982 137 Design_4 26.11 5438 1213 168 Design_5 113.68 5388 1865 249 Design_6 178.56 5789 3914 613 Design_6a 173.60 5794 2822 405 Design_7 75.69 6322 1929 259 Design_8 98.63 6412 1831 243

[0461] These results were completely different than Wu et al.

observed in the design of similar antibody-TCR chimeras (Wu et al. 2015, supra): Their “cross-orientation” designs showed low expression. His “normal orientation” drawings didn't even express.

[0462] To confirm that the expressed proteins folded correctly and maintained their original function, we tested their binding on Jurkat CD3 positive cells.

FACS connections of all samples were performed in three different concentrations: 5.0, 0.4 and 0.032 nm. The original T3 wild-type antibody was used as a positive control. Dose-dependent CD3 binding data was listed in column 3-5 in Table 24. Design_2, Design_6 and Design_6a showed better binding capacity, comparable to native T3 antibody. It is interesting that all three of these constructions were the three best formats expressed in mammalian cells. This strong correlation suggested that the level of expression or binding may result from the same molecular origin, that is, the compatibility between the variable domain of the antibody and the domains in the TCR, which required careful design of the components, such as the junction and interchain disulfide bond etc.

[0463] Based on the level of expression and the liaison activity, Design_2 was selected as the final format to proceed.

EXAMPLE 3: Deglycosylation

[0464] Post-translational modifications (PTM), such as N-glycosylation sites in an antibody, can cause heterogeneity of proteins, becoming a challenge in the development stages. Therefore, an attempt was made to remove the N-glycosylation sites in the TCR constant region. There are a total of four N-glycosylation sites found in the TCR constant region. One is in CBeta (N69, see SEQ ID NO: 244) and the other three are in CAlfa (N34, N68 and N79, see SEQ ID NO: 241). The expression data from the present disclosure suggested that these sites, especially the CAlfa sites, were in fact strongly glycosylated when the molecule was expressed in the mammalian cell.

[0465] All glycosylation sites in Design_2 have been removed by replacing the four Asn residues with Gln or Ala (see Design_2-QQQQ or –AAAA, see Table 25). Although this strategy is very general in protein modification, it has been reported that Gln / Ala mutations can affect the level of expression of chimeric TCR / antibody proteins (Wu et al., 2015, supra). To mitigate this risk, residues of pre-TCR (N68S in CAlfa) and monkey TCR (N79 in CAlfa, N69E in CBeta) were also used in the corresponding positions

(see Design_2-QSKE and -ASKE) (see Table 25). In addition, it has been reported that there may be an atypical glycosylation site in CAlfa (N 6 1) (Wollscheid et al., Nature Biotechnology, 27 (4), pp. 378-386 (2009) "Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. "WollscheidB., Bausch- Fluck D., Henderson C., O'Brien R., Bibel M., Schiess R., Aebersold R., Watts JD, Nat.

Biotechnol. 27: 378-386 (2009) [PubMed] [Europe PMC]). Therefore, this residue has also been changed to Gln (see Design_2-QQQQQ, see Table 25).

All mutants were expressed in Expi293 for further testing.

TABLE 25. Supernatant expression levels of all deglycosylation designs Supernatant expression level SEQ ID NO: Sample (µg / mL)) (HC-CBeta / LC-CAlfa) 13/12 (IgG1) Design_2-QQQQ 334 , 39 12/21 (IgG4) Design_2-AAAA 414,58 15/14 (IgG1) Design_2-QSKE 311,48 17/16 (IgG1) Design_2-ASKE 107,89 17/18 (IgG1) Design_2-QQQQQ 213,31 20/19 (IgG1) TABLE 26 Components of deglycosylation designs Domains from the N-terminus to the C-terminus and their SEQ ID NOs. Complex Third Name and SEQ First Variable Domain or Domain Domain ID Domain NOs. of the heavy chain according to the constant of the junction + Dimerization chain: of the antibody (VH domain of TCR (C1 or hinge (D) or VL) junction (CJ) C2) (CJ ') Design_2-QQQQ CBeta (S56C) VH (CD3) HCJ2 CJ'1G1 FcG1 (IgG1) HC (N69Q) SEQ ID NO: 13 SEQ ID NO: 300 SEQ ID NO: 50 SEQ ID NO: 53 SEQ ID NO: 53 SEQ ID NO: 302 CAlpha (T49C) Design_2-QQQQ VL (CD3) LCJ2 (N34Q + N68Q + (IgG1) LC N79Q) SEQ ID NO: 12 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 44 Design_2-QQQQ CBeta (S56C) VH (CD3) HCJ2 CJ'1G4 FcG4 (IgG4) HC (N69Q) SEQ ID NO: 21 SEQ ID NO: 300 SEQ ID NO: 50 SEQ ID NO: 34 SEQ ID NO: 54 SEQ ID NO: 303 CAlpha (T49C) Design_2-QQQQ VL (CD3) LCJ2 ( N34Q + N68Q + (IgG4) LC N79Q) SEQ ID NO: 12 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 44

Domains from the N-terminus to the C-terminus and their SEQ ID NOs. Complex Third Name and SEQ First Variable Domain or Domain Domain ID Domain NOs. of heavy chain according to constant of junction + Dimerization chain: of the antibody (VH domain of TCR (C1 or hinge (D) or VL) junction (CJ) C2) (CJ ') Design_2-AAAA CBeta (S56C) VH (CD3) HCJ2 CJ'1G1 FcG1 (IgG1) HC (N69A) SEQ ID NO: 15 SEQ ID NO: 300 SEQ ID NO: 35 SEQ ID NO: 53 SEQ ID NO: 53 SEQ ID NO: 302 CAlpha (T49C) Design_2-AAAA VL (CD3) LCJ2 (N34A + N68A + (IgG1) LC N79A) SEQ ID NO: 14 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 45 Design_2-QSKE CBeta (S56C) VH (CD3) HCJ2 CJ'1G1 FcG1 (IgG1) HC (N69E) SEQ ID NO: 17 SEQ ID NO: 300 SEQ ID NO: 50 SEQ ID NO: 36 SEQ ID NO: 53 SEQ ID NO: 302 CAlpha (T49C) Design_2-QSKE VL (CD3) LCJ2 ( N34Q + N68S + (IgG1) LC N79K) SEQ ID NO: 16 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 46 Design_2-ASKE CBeta (S56C) VH (CD3) HCJ2 CJ'1G1 FcG1 (IgG1) HC ( N69E) SEQ ID NO: 17 SEQ ID NO: 300 SEQ ID NO: 50 SEQ ID NO: 36 SEQ ID NO: 53 SEQ ID NO: 302 CAlpha (T49C) Design_2-ASKE VL (CD3) LCJ2 (N34A + N68S + (IgG1 ) LC N79K) SEQ ID NO: 18 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 47 Design_2- CBeta (S56C) QQQQQ (IgG1) VH (CD3) HCJ2 CJ'1G1 FcG1 (N69Q)

HC SEQ ID NO: 20 SEQ ID NO: 300 SEQ ID NO: 50 SEQ ID NO: 34 SEQ ID NO: 53 SEQ ID NO: 302 Design_2- CAlpha (T49C) QQQQQ (IgG1) VL (CD3) LCJ2 (N34Q + N68Q + LC N79Q + N61Q) SEQ ID NO: 19 SEQ ID NO: 301 SEQ ID NO: 52 SEQ ID NO: 48

[0466] The amounts of expression in the supernatants were estimated by Q-ELISA and shown in Table 25. Interestingly, only one of our deglycosylation designs slightly decreased the level of expression. Simple Gln or Ala mutations had no negative effects on the unreduced gel (Figure 4), and a 150 kd band was observed. In the reducing gel (Figure 4), the 25 kd band was observed.

Both indicate the successful removal of glycosylation in the light chain and the heavy chain. Muteins with the removal of N-glycans in the TCR constant region were tested for binding to CD3. Figure 5 illustrates the different muteins that bind to Jurkat CD3 + cells. The mutein curves only shifted slightly to the right compared to the wild-type T3 antibody, which may be due to the detection of antibodies being more sensitive to human IgG than to chimera. The maximum connection has not been changed. In general, most deglycosylation designs did not exhibit obvious differences in expression or binding. Design_2-QQQQ was chosen as the design for further studies.

[0467] In a similar study by Wu et al., (Wu et al. 2015, supra), they carried out deglycosylation mutations in their “cross-orientation” formats, since their “normal orientation” formats do not express.

EXAMPLE 4: WuXiBody design based on pre-alpha / beta TCR

[0468] The pre-cell antigen receptor (pre-TCR), expressed by immature thymocytes, plays a central role in the early development of T cells.

The pre-TCR has a regular beta chain, but a special pre-alpha chain, with only a constant region available, whose sequence and structure are quite different from those of the regular alpha chain. As the constant region of the regular TCR is compatible with the variable region of the antibody, it was expected that Pre-TCR (see PDB 3OF6, SEQ ID NO: 246) would also assist in the development of chimeric proteins. The antibody designs were shown in Table 27.

TABLE 27. Design of chimeras based on pre-alpha / beta TCR for WuXiBody Handle Handle Orientation Conjunction Conjunction ’

FG DE Pre- Conjunction'_1 Normal Conjunction_A Native Native TCR_Design_A (IgG1, IgG4) Pre- Conjunction'_1 Normal Conjunction_B Native Native TCR_Design_B (IgG1, IgG4) Pre- Conjunction'_2 Crossed Conjunction_C Native Native TCR_Design_C (IgG1, IgG4) Pre- '_2 Crusade Conjunction_D Native Native TCR_Design_D (IgG1, IgG4)

[0469] In total, ten chimeric constructions were developed combining these parameters, as listed in Table 28.

TABLE 28. Correspondence of the chimeric pre-TCR chimeric antibody design in the form of IgG1

Drawings in Table SEQ ID NOs: Sequence File 27 (HC / LC) Design_1_Pre_TCR_ Conjunction'1 65/64 Design_2_Pre_TCR_Conjunction'_1_Cys10 67/66 Design_3_Pre_TCR_Conjunction'_1_Cys11 69/68 Design_4_Pre_TCR_Conjunction'_1_C_12_Design__1_Cys12/68 Design_6_Pre_TCR_Conjunction'_1_Cys14 Design_7_Pre_TCR_Conjunction'_1_Cys15 72/71 73/71 75/74 Design_8_Pre_TCR_Conjunction'_1_Cys1_4L4T_1 Design_9_Pre_TCR_Conjunction'_1_Cys2_4L4T_2 Design_10_Pre_TCR_Conjunction'_1_Cys4 75/76 78/77 137/136 TCR_Design_5_crossed_1 Pre-Pre-Pre-TCR_Design_C TCR_Design_6_crossed_1 137/139 Pre-TCR_Design_5_crossed_2 138 / 136 Pre-TCR_Design_D Pre-TCR_Design_6_crossed_2 138/139

TABLE 29. Design components of the chimeric pre-TCR chimeric antibody in the form of IgG1 Domains from the N-terminus to the C-terminus and their SEQ ID NOs.

Domain Third Complex name and First or Domain variable Domain of SEQ ID NOs. of the second constant of the heavy chain junction + Dimerization chain: TCR domain (C1 or antibody hinge (D) junction (CJ) C2) (VH or VL) (CJ ') Design_1_Pre_TCR_ VH (CD3) HCJB CBeta (N69Q) CJ' 1G1 FcG1 Conjunction'1 HC SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 65 SEQ ID NO: 302 300 NO: 50 NO: 84 NO: 53 Design_1_Pre_TCR_ CPreAlpha VL (CD3) LCJB Conjunction'1 LC (N50Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 64 301 NO: 309 NO: 83 Design_2_Pre_TCR_C Cbeta onjunction'_1_Cys10 VH (CD3) HCJB (S76C) (N69Q CJ'1G1 FcG1 HC) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 67 SEQ ID NO: 302 300 NO: 50 NO: 319 NO: 53 Design_2_Pre_TCR_C CPreAlpha onjunction'_1_Cys10 VL (CD3) LCJB (Y59C) (N50Q LC) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 66 301 NO: 309 NO: 311 Design_3_Pre_TCR_C Cbeta onjunction'_1_Cys11 VH (CD3) HCJB (F13C) (N69Q CJ'1G1 FcG1 HC) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 69 SEQ ID NO: 302 300 NO: 50 NO: 320 NO: 53 Design_3_Pre_TCR_C CPreAlpha onjunction'_1_Cys11 VL (CD3) LCJB (A13C) (N50Q LC) SEQ ID NO: SEQ ID SE Q ID SEQ ID NO: 68 301 NO: 309 NO: 312 Design_4_Pre_TCR_C Cbeta onjunction'_1_Cys12 VH (CD3) HCJB (S16C) (N69Q CJ'1G1 FcG1 HC) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 70 SEQ ID NO: 302 300 NO: 50 NO: 321 NO: 53

Domains from the N-terminus to the C-terminus and their SEQ ID NOs.

Domain Third Complex name and First or Domain variable Domain of SEQ ID NOs. of the second constant of the heavy chain junction + Dimerization chain: TCR domain (C1 or antibody hinge (D) junction (CJ) C2) (VH or VL) (CJ ') Design_4_Pre_TCR_C CPreAlpha onjunction'_1_Cys12 VL (CD3) LCJB (A13C ) (N50Q LC) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 68 301 NO: 309 NO: 312 Design_5_Pre_TCR_C Cbeta onjunction'_1_Cys13 VH (CD3) HCJB (S16C) (N69Q CJ'1G1 FcG1 HC) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 70 SEQ ID NO: 302 300 NO: 50 NO: 321 NO: 53 Design_5_Pre_TCR_C CPreAlpha onjunction'_1_Cys13 VL (CD3) LCJB (S11C) (N50Q LC) SEQ ID NO: SEQ ID SEQ SEQ ID ID NO: 71 301 NO: 309 NO: 313 Design_6_Pre_TCR_C Cbeta onjunction'_1_Cys14 VH (CD3) HCJB (A18C) (N69Q CJ'1G1 FcG1 HC) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 72 SEQ ID ID NO: 302 300 NO: 50 NO: 322 NO: 53 Design_6_Pre_TCR_C CPreAlpha onjunction'_1_Cys14 VL (CD3) LCJB (S11C) (N50Q LC) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 71 301 NO: 309 NO: 313 Design_7_Pre_TCR_C Cbeta onjunction'_1_Cys15 VH (CD3) HCJB (E19C) (N69Q CJ'1G1 FcG1 HC) SEQ ID NO: SEQ I D SEQ ID SEQ ID SEQ ID NO: 73 SEQ ID NO: 302 300 NO: 50 NO: 323 NO: 53 Design_7_Pre_TCR_C CPreAlpha onjunction'_1_Cys15 VL (CD3) LCJB (S11C) (N50Q LC) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 71 301 NO: 309 NO: 313 Design_8_Pre_TCR_C Cbeta onjunction'_1_Cys1_4L VH (CD3) HCJB (S56C) (N69Q CJ'1G1 FcG1 4T_1 HC) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 75 SEQ ID ID NO: 302 300 NO: 50 NO: 34 NO: 53 Design_8_Pre_TCR_C CPreAlpha onjunction'_1_Cys1_4L VL (CD3) LCJB (S62C) (N50Q 4T_1 LC) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 74 301 NO: 309 NO : 314 Design_9_Pre_TCR_C Cbeta onjunction'_1_Cys2_4L VH (CD3) HCJB (S56C) (N69Q CJ'1G1 FcG1 4T_2 HC) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 75 SEQ ID NO: 302 300 NO: 50 NO: 34 NO: 53 Design_9_Pre_TCR_C CPreAlpha onjunction'_1_Cys2_4L VL (CD3) LCJB (T65C) (N50Q 4T_2 LC) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 76 301 NO: 309 NO: 315

Domains from the N-terminus to the C-terminus and their SEQ ID NOs. Domain Third Complex name and First or Domain variable Domain of SEQ ID NOs. of the second constant of the heavy chain junction + Dimerization chain: TCR domain (C1 or antibody hinge (D) junction (CJ) C2) (VH or VL) (CJ ') Design_10_Pre_TCR_ Cbeta Conjunction'_1_Cys4 VH (CD3) HCJB (A11C ) (N69Q CJ'1G1 FcG1 HC) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 78 SEQ ID NO: 302 300 NO: 50 NO: 324 NO: 53 Design_10_Pre_TCR_ CPreAlpha Conjunction'_1_Cys4 VL (CD3) LCJB ( I16C) (N50Q)

LC SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 77 301 NO: 309 NO: 316 Pre-CPre-Alpha TCR_Design_5_crosse VH (CD3) HCJC (S11C) (N50Q CJ'2G1 FcG1 d_1 HC) SEQ ID SEQ ID SEQ ID NO: SEQ ID NO: 137 SEQ ID NO: 300 SEQ ID NO: 302 NO: 132 NO: 313 134 Pre-Beta (N69Q, TCR_Design_5_crosse VL (CD3) LCJC S16C) d_1 LC SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID NO: 136 301 NO: 50 NO: 321+ 306 Pre-CPreAlpha TCR_Design_6_crosse VH (CD3) HCJC (S11C) (N50Q CJ'2G4 FcG4 d_1 HC) SEQ ID SEQ ID SEQ ID NO: SEQ ID NO: 137 SEQ ID NO: 300 SEQ ID NO: 303 NO: 132 NO: 313 134 Pre- Cbeta (N69Q, TCR_Design_6_crosse VL (CD3) LCJC A18C) d_1 LC SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 139 301 NO: 50 NO: 322+ 306 Pre- CPreAlpha TCR_Design_5_crosse VH (CD3) HCJD (S11C) (N50Q CJ'2G1 FcG1 d_2 HC) SEQ ID SEQ ID SEQ ID NO: SEQ ID NO: 138 SEQ ID NO: 300 SEQ ID NO: 302 NO: 133 NO: 313 134 Pre- Cbeta (N69Q, TCR_Design_5_crosse VL (CD3) LCJC S16C) d_2 LC SEQ ID NO: SEQ ID SEQ ID NO: SEQ ID NO: 136 301 NO: 50 NO: 321+ 306 Pre- CPreAlpha TCR_Design_6_crosse VH (CD3) HCJD (S11C) (N5 0Q CJ'2G1 FcG1 d_2 HC) SEQ ID SEQ ID SEQ ID NO: SEQ ID NO: 138 SEQ ID NO: 300 SEQ ID NO: 302 NO: 133 NO: 313 134 Pre-Cbeta (N69Q, TCR_Design_6_crosse VL (CD3) LCJC A18C) d_2 LC SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 139 301 NO: 50 NO: 322 + 306

[0470] The experience learned in Examples 1-3 suggested that the "normal orientation" and the junction domain with more TCR residues were more suitable for producing good chimeric proteins. Thus, the same strategy was adopted and the light and heavy chain junction domains, as shown in Table 3 and Table 4, were developed. Unlike the regular alpha chain, there is only one glycosylation site (N50) in the pre-alpha chain of the TCR, which has been mutated to the Gln residue (see SEQ ID NO: 247). The entire heavy chain with the beta constant region was the same as Design_2 in Table 22, with the N-glycosylation site (N69) replaced by the Gln residue (see SEQ ID NO: 244).

[0471] The third junction domains in normal orientation were developed identically to that shown in Table 7, and the third junction domains in cross orientation were developed identically to that present in Table 8 (chimeric antibodies based on alpha / beta TCR .

[0472] The pre-TCR has no native interchain disulfide bond above the third junction domain. Similar to the construction work performed on the regular TCR, we rationally introduced the disulfide bond at the beta and pre-alpha interface in the constant region to improve the stability of the chimeric protein (see Table 11). All interface residues in the crystalline structure of the pre-TCR (APO 3OF6) were examined and the list of interchain pairs was obtained, whose carbon atoms of CAlfa and CBeta were within 7 Å and 5 Å, respectively (see Table 11) .

Each identified pair was then replaced by Cys residues and mutein was expressed in Expi293 cells.

EXAMPLE 5: Chimeric antibody designs based on gamma / delta TCR

[0473] TCRs composed of gamma and delta chains are less common, but the heterodimeric nature of the protein may also help develop a new chimeric format. Following the same strategy and procedure that were validated in Example 1, we carried out new chimeric designs that used the delta-gamma TCR constant region to replace the corresponding antibody region. The structure of the delta-gamma TCR (PDB 4LFH, see SEQ ID NO: 249 and 252) was used to facilitate the alignment of the sequence guided by the structure between the antibody and the TCR.

[0474] Table 5 and Table 6 listed junction domains developed for “normal orientation” and “cross orientation”, respectively. The corresponding IgG1 and IgG4 junction domains from different orientations were shown in Table 9 and Table 10. The structure of the delta-gamma TCR is more similar to the antibody than the structure of the alpha-beta TCR. No additional FG and DE handle designs were performed. The N-glycosylation sites (N65 in the range, and N16 and N79 in the Delta, see SEQ ID NO: 250) were all removed by Gln (Q) substitutions. The disulfide bond of the contact interface was modified based on the same strategy introduced in Example 4.

TABLE 30. TCR / Chimeric antibody design First and second Third domain Orientation FG loop DE junction junction domains Conjunction'_3 dg_Design_1 Normal Conjunction_4 Native Native (IgG1, IgG4) Conjunction'_3 dg_Design_2 Normal Conjunction_5 Native Native (IgG1, IgG4) Conjunction'_4 dg_Design_3 Crusade Conjunction_6 Native Native (IgG1, IgG4) Conjunction'_4 dg_Design_4 Crusade Conjunction_7 Native Native (IgG1, IgG4)

[0475] A total of thirteen chimeric constructs were developed combining these parameters, as listed in Table 31.

TABLE 31. Correspondence of the TCR / chimeric antibody design to Gamma / CDelta Drawings in the Table Construction of Design SEQ ID NOs. in HC / LC 30 IgG1 dg_Design_1 dg_Design_1 dg_Design_2 108/107 106/105 86/85 dg_Design_2_no_Glyco dg_Design_2 dg_Design_2_hypeCys1_no_Glyco dg_Design_2_hypeCys2_no_Glyco 90/89 92/91 94/93 dg_Design_2_hypeCys3_no_Glyco dg_Design_2_Cys2_no_Glyco dg_Design_2_Cys1_no_Glyco 96/95 98/97 99/95 dg_Design_2_Cys3_no_Glyco dg_Design_2_Cys4_no_Glyco 101/100 dg_Design_2_Cys5_no_Glyco 101/102 dg_Design_3 dg_crossed_Design_1 110/109 dg_Design_4 dg_crossed_Deisgn_2 112/111 TABLE 32. Components of the TCR / chimeric antibody design for gamma / CDelta Domains from the N-terminal to the C-terminal and their SEQ ID NOs. First Third Domain or Domain Name of the complex and domain variable of Second Domain contained in SEQ ID NOs. chain: heavy chain junction + Dimerization domain of TCR (C1 or antibody hinge (D) junction (CJ) C2) (VH or VL) (CJ ') dg_Design_1 HC VH (CD3) HCJ4 CGamma CJ'3G1 FcG1 SEQ ID NO : SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 108 300 NO: 117 NO: 113 NO: 121 NO: 302 dg_Design_1 LC VL (CD3) LCJ4 CDelta SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 107 301 NO : 119 NO: 310 dg_Design_2 HC VH (CD3) HCJ5 CGamma CJ'3G1 FcG1 SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 106 300 NO: 118 NO: 113 NO: 121 NO: 302 dg_Design_2 LC VL (CD3) LCJ5 CDelta SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 105 301 NO: 120 NO: 115 dg_Design_2_no_Glyco CGamma VH (CD3) HCJ5 CJ'3G1 FcG1 HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 86 300 NO: 118 NO: 114 NO: 121 NO: 302 CDelta dg_Design_2_no_Glyco VL (CD3) LCJ5 (N16Q + N79Q

LC) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 85 301 NO: 120 NO: 116 CGamma dg_Design_2_hypeCys1 VH (CD3) HCJ5 (T12C) CJ'3G1 FcG1 _no_Glyco HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 90 300 NO: 118 NO: 333 NO: 121 NO: 302 CDelta dg_Design_2_hypeCys1 VL (CD3) LCJ5 (N16C) _no_Glyco LC (N79Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 89 301 NO: 120 NO: 325 CGamma dg_Design_2_hypeCys2 VH (CD3) HCJ5 (Q57C) CJ'3G1 FcG1 _no_Glyco HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 92 300 NO: 118 NO: 334 NO : 121 NO: 302 CDelta dg_Design_2_hypeCys2 (V50C) VL (CD3) LCJ5 _no_Glyco LC (N16Q + N79Q)

Domains from the N-terminus to the C-terminus and their SEQ ID NOs.

First Third Domain or Domain Name of the complex and domain variable of Second Domain contained in SEQ ID NOs. chain: heavy chain junction + Dimerization domain of TCR (C1 or antibody hinge (D) junction (CJ) C2) (VH or VL) (CJ ') SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 91 301 NO : 120 NO: 326 CGamma dg_Design_2_hypeCys3 VH (CD3) HCJ5 (M62C) CJ'3G1 FcG1 _no_Glyco HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 94 300 NO: 118 NO: 335 NO: 121 NO: 302 CDelta dg_Design_2_hypeCys3 (D46C) VL (CD3) LCJ5 _no_Glyco LC (N16Q + N79Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 93 301 NO: 120 NO: 327 CGamma dg_Design_2_Cys2_no_ HCV (CD3) ) CJ'3G1 FcG1 Glyco HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 96 300 NO: 118 NO: 336 NO: 121 NO: 302 CDelta dg_Design_2_Cys2_no_ VL (CD3) LCJ5 (F12C) (N16Q Glyco LC + N79Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 95 301 NO: 120 NO: 328 CGamma dg_Design_2_Cys1_no_ VH (CD3) HCJ5 (F14C) CJ'3G1 FcG1 Glyco HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 98 300 NO: 118 NO: 337 NO: 121 NO: 302 CDelta dg_Design_2_Cys1_no_ (M14C) VL (CD3) LCJ5 Glyco LC (N16Q + N79 Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 97 301 NO: 120 NO: 329 CGamma dg_Design_2_Cys3_no_ VH (CD3) HCJ5 (E20C) CJ'3G1 FcG1 Glyco HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 99 300 NO: 118 NO: 338 NO: 121 NO: 302 CDelta dg_Design_2_Cys3_no_ VL (CD3) LCJ5 (F12C) (N16Q Glyco LC + N79Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 95 301 NO: 120 NO: 328 CGamma dg_Design_2_Cys4_no_ VH (CD3) HCJ5 (A19C) CJ'3G1 FcG1 Glyco HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 101 300 NO: 118 NO: 339 NO: 121 NO: 302 CDelta dg_Design_2_Cys4_no_ VL (CD3) LCJ5 (F87C) (N16Q Glyco LC + N79Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 100 301 NO: 120 NO: 330

Domains from the N-terminus to the C-terminus and their SEQ ID NOs. First Third Domain or Domain Name of the complex and domain variable of Second Domain contained in SEQ ID NOs. chain: heavy chain junction + Dimerization domain of TCR (C1 or antibody hinge (D) junction (CJ) C2) (VH or VL) (CJ ') CGamma dg_Design_2_Cys5_no_ VH (CD3) HCJ5 (A19C) CJ'3G1 FcG1 Glyco HC (N65Q) SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 101 300 NO: 118 NO: 339 NO: 121 NO: 302 CDelta dg_Design_2_Cys5_no_ (E88C) LCJ5 Glyco LC (N16Q + N79Q)

SEQ ID SEQ ID SEQ ID NO: 102 NO: 120 NO: 331 dg_crossed_Design_1 VH (CD3) HCJ6 CDelta CJ'4G1 FcG1

HC SEQ ID NO: SEQ ID SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 110 300 NO: 123 332 NO: 127 NO: 302 dg_crossed_Design_1 VL (CD3) LCJ6 CGamma

LC SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 109 301 NO: 125 NO: 340 dg_crossed_Design_2 VH (CD3) HCJ7 CDelta CJ'4G1 FcG1

HC SEQ ID NO: SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 112 300 NO: 124 NO: 332 NO: 127 NO: 302 dg_crossed_Design_2 VL (CD3) LCJ7 CGamma

LC SEQ ID NO: SEQ ID SEQ ID SEQ ID NO: 111 301 NO: 126 NO: 340 EXAMPLE 6: Incorrect antibody heavy chain matching tests

[0476] One of the challenges in the production of bispecific antibody in IgG type format is the uncontrolled mismatch of light and heavy chains. We evaluated whether the CH1 and CL domains replaced by beta and alpha TCR can join the heavy and normal IgG light chains when they were c-expressed in a single host cell.

[0477] In addition to the anti-CD3 T3 antibody, we have also developed a U4 monoclonal antibody that targets the B lymphocyte antigen, CD19. To check the likelihood that the light and heavy chains of the two native antibodies could be mismatched, the light and heavy pairs of T3 and U4 were purposely switched (T3_light-U4_pesada, T3_pesada-U4_leve) and coexpressed in Expi293 cells. The same study using T3 modified with TCR was also performed as a side-by-side comparison. Figures 6A-6B presented the data of SDS-

PAGE of proteins in both IgG1 and IgG4. For switched pairs using native antibodies, the 150 kd band on the non-reducing PAGE and the 50 kd and 23 kd bands on the reducing PAGE clearly confirmed the incorrectly paired IgG protein assembly. However, after the introduction of T3 modified with TCR, the 150 kd bands were no longer observed in the gel, indicating that none of the non-cognate pairs can join the antibody-like molecule. These data confirmed that our modified Fab with developed TCR can effectively prevent incorrect pairing of non-cognitive chains.

EXAMPLE 7: Production and characterization of the Fab-TCR chimera

[0478] To ensure that the TCR-modified Fab antibody can be used to develop the bispecific antibody, truncated Fab fragments in two positions were constructed. Figure 8 illustrates that TCR modified T3 Fabs with removed N-glycan have been successfully expressed and purified (T3-Fab- Design_2.his1 (SEQ ID NO: 30/12) and T3-Fab-Design_2.his2 (SEQ ID NO: 12/31)). Its ability to bind to CD3 was also evaluated in Jurkat CD3 + cells and compared to the monovalent wild type T3. Figure 9 showed that the chimeric Fab and monovalent T3 had qualitatively similar binding behaviors. The deviations may result from the difference in protein detection methods with the His and Fc markers.

EXAMPLE 8: Generation and characterization of bispecific antibody with TCR-based knob-into-holes

[0479] After the successful fusion of the constant domain of the TCR into the monospecific antibody T3 and confirmation that the new format can effectively prevent the chain from being incorrectly paired with the U4 antibody, we proceed to build bispecific formats.

[0480] T3 modified with TCR and wild-type U4, with “knob-into-holes” mutations used in the CH3 Fc domain, were coexpressed from cells

Expi293. Mutations for “knob-into-holes” were made in S139C and T151W in the CH3 domain (SEQ ID NO: 295, knob) of T3 and Y134C, T151S, L153A and Y192V in the CH3 domain (SEQ ID NO: 296, hole) of U4 in the IgG1 isotype. Alternatively, the “knob-into-holes” mutations were made in S136C and T148W in the CH3 domain (SEQ ID NO: 298, knob) of T3 and Y131C, T148S, L150A and Y189V in the CH3 domain (SEQ ID NO: 299, hole ) of U4 in the IgG4 isotype. Figures 7A-7B show the SDS-PAGE data of proteins produced in IgG1 and IgG4, after purifications. The yield after the first purification step in protein A reached 125 mg / L and 173.7 mg / L, for IgG1 and IgG4, respectively. The correct molecular weight, that is, the bands around 150 kd in non-reduced gel, as well as the bands around 50 and 25 kd in reducing gel, were all clearly observed. The purified samples were later inspected on SEC-HPLC. The purity of IgG1 and IgG4 reached 98.63% and 100%. The data indicated that the IgG type molecules, both IgG1 and IgG4, were well expressed and assembled. These new bispecific formats involved with TCR were referred to as 'E17-Design_2-QQQQ' (SEQ ID NO: 12/22/24/23 for IgG1 and SEQ ID NO: 12/25/26/23 for IgG4).

[0481] Although the expected molecular weight was observed for the bispecific antibody developed, it was necessary to verify that each arm maintained its original binding capacity to the individual cognate antigen. As for each target, E17-Design_2 was a monovalent binder, we also built the monovalent version of native T3 and native U4 to make side-by-side comparisons. Figure 10A and Figure 10B show the results of FACS binding of the bispecific antibody developed for Jurkat CD3 + cells and Ramos CD19 + cells, respectively. The T3 arm modified with TCR showed moderate loss of binding compared to the wild type T3, but IgG4 was better than IgG1 and close to the native protein. The connection of the U4 arm was not reduced by the adjacent modified T3 arm. It showed similar binding to the original U4 antibody in monovalent form. But, interestingly, this time IgG1 performed better than IgG4. It is not clear why the isotype is important in maintaining the monovalent bond. Factors such as stability of the TCR constant region, selection of third junction domain designs or interactions between the two Fab arms can result in the observed phenomena.

[0482] Monovalent bonds of the TCR-modified bispecific format for CD3 and CD19 were both reduced compared to their bivalent parental antibodies. Activation of T cells through binding to CD3 is known to be quite sensitive. Strong stimuli for T cells can cause side effects.

Therefore, the relatively weak binding to CD3 was probably acceptable and even desired for safety reasons. However, poor binding to CD19 can directly affect its ability to kill B cells driven by bispecific antibodies and thus reduce the drug's effectiveness. To confirm the importance of binding to CD19 and to test the universality of our chimeric designs, we built other bispecific constructions called “F16-Design_2-QQQQ” in IgG4, in which the developed T3 arm was still monovalent, but the U4 arm was bivalent.

[0483] The new construction was expressed and purified, and the bonding experiment was carried out directly. Figures 11A-11B showed their FACS binding data in relation to the previously developed E17 and two parental antibodies T3 and U4. It is interesting that F16-Design_2-QQQQ improved the binding to CD3 and CD19 (SEQ ID NO: 25/12/27/23 in the order HC / LC (anti-CD3) / HC / LC (anti-CD19)). Its binding to CD19 (SEQ ID NO: 27/23) was comparable to that of wild type U4 antibody. The data confirmed that our chimeric T3 design can be applied to different bispecific formats.

EXAMPLE 9: In vitro tumor cell death test directed by bispecific antibodies

[0484] The functional in vitro assay was performed to verify the activity of the bispecific format developed in the death of malignant B cells with T cell involvement. The E17 construct was tested first. Parental monospecific antibodies T3 and U4 were used as a negative control. Figure 12 illustrates the dose-dependent cell death function of this bispecific E17 format.

E17-IgG4 (EC50 = 57 pM) was more potent than E17-IgG1 (EC50 = 624 pM). In order to improve cell death activity, the F16 format, which had two CD19 binding sites, was also compared to E17. As shown in Figure 13, compared to E17 (EC50 = 17.7 pM), the power of F16 (EC50 = 5.5 pM) was improved 3 times. The data confirmed that CD19 binding affected the effect of cell death. An irrelevant human IgG4 antibody was used as a negative control.

EXAMPLE 10: Characterization by mass spectrometry

[0485] To confirm that the bispecific antibody produced had the correct assembly, we characterized the molecule E17-Design_2-QQQQ by mass spectrometry. The theoretical molecular weight differences between the two heavy chains and the two light chains are approximately 4000 Da and 500 Da, respectively. Figure 14A illustrates the protein spectra in a non-reducing condition. The peak at 148180 Da was the expected weight of the bispecific antibody molecule assembled correctly. No other peak observed indicates that the “knob-into-holes” mutations in the Fc region, as well as our CH1 / CL region replaced by TCR, worked adequately in the pairing of the four desired chains.

It is noteworthy that the non-reduced mass spectra cannot help to distinguish the correct assembled bispecific antibody from IgG that has both light chains incorrectly paired. However, Example 4 indicates that the heavy and light chains incorrectly paired do not express or assemble, which eliminated the possibility of mismatching errors of two pairs of heavy and light chains.

[0486] In non-reducing conditions (see Figure 14A), there was a peak at 149128 Da, which is about 947 Da more than the calculated weight of the molecule. A mass spectrometry analysis was also performed using the protein in reduced condition. Figure 14B showed that there was indeed a peak of 948 Da distant from the chimeric light chain VL-CAlfa, indicating modifications of O-glycan (GlcNAc + Hex + 2 * NeuAc) in the light chain.

EXAMPLE 11: Thermal stability tests

[0487] We further tested and compared the thermal stability of bispecific antibodies developed in both IgG1 and IgG4 by measuring the protein's Tm fusion temperature using differential scanning fluorimetry (DSF). Native monospecific T3 and T3 modified with TCR (Design_2 and Design_2-QQQQ) were used as controls.

[0488] Table 33 lists the measured values of Ton and Tm of the new constructions. In general, all molecules showed reasonable thermal stability. IgG1-type molecules were more stable than IgG4-type molecules. The Tm value of the native T3 antibody was 74 ° C. The chimeric antibody and TCR proteins had a relatively low Tm of about 60 ° C, suggesting that CBCR-CAlfa of TCR may be less resistant to elevated temperatures, compared to the CH1-CL of the normal antibody. This is consistent with what has been reported from Wu's study (Wu et al. 2015, supra), and it has been suggested that the CAlfa domain is less stable than CBeta (Toughiri et al. MAbs, 862 (July), pp. 1276-1285 (2016)).

[0489] The mutations that removed N-glycosylation in the TCR constant region did not affect the thermal stability of the chimeric protein. Our bispecific antibody E17-Design_2-QQQQ showed Tm similar to that of Design_2-QQQQ, and lower Tm than native T3.

TABLE 33. Thermostability of the developed chimeric and bispecific antibody, measured by differential scanning fluorimetry (DSF) Protein Name Concentration Ton Th 1 (Tm) Th 2 HC / LC (anti-CD3) / HC / LC Isotype pI Purity (mg / ml) (℃) (℃) (℃) (anti-CD19) T3 IgG1 2.7 57 74.2 na 8.31 99.41% Design_2 (SEQ ID NO: IgG1 1.6 46 59.3 na 6, 07 93.09% 4/3/4/3) Design_2-QQQQ IgG1 1.1 45 59.1 na 6.07 99.03% (SEQ ID NO: 12/13/13/12) E17-Design_2-QQQQ (SEQ IgG1 0.3 49 61.9 76.2 7.29 98.63% ID NO: 12/22/24/23) T3 IgG4 1.4 53 65 73.2 8.24 96.06% Design_2- QQQQ IgG4 0.9 45 58.4 na 5.7 96.05% (SEQ ID NO: 12/21/21/12) E17-Design_2-QQQQ (SEQ IgG4 0.8 47 60.2 72.7 6, 4 100% ID NO: 12/25/26/23) EXAMPLE 12: Materials and Methods Modeling T3 Antibody Fv Homology

[0490] The Fv structural model of the antibody was built based on its Fv amino acid sequences using the Discovery Studio software (BIOVIA). The light and heavy chain sequences were noted first in the Kabat numbering to identify three CDRs, as well as the structure of each chain.

Each segment (CDR or structure) was then searched in the BLAST antibody database with sequences of all antibody structures in the PDB. The structure of the most corresponding sequence, if it has high resolution and low factor B, was used to build the homology model. All modeled segments were assembled to build the light and heavy chain structural model. The relative orientation between two modeled chains was predicted by taking the angle of the antibody structure that had the most similar complete sequence. All the work of visualization and molecular analysis was performed using the PyMOL software (Schrödinger).

Vector constructions

[0491] The VL, VH, Ck, CH1 genes were amplified by PCR from existing internal DNA models. The CAlfa and CBeta genes were synthesized by

Genewiz Inc. Native or chimeric light chain genes were inserted into a linearized vector containing a CMV promoter and a kappa signal peptide. The DNA fragments of VH-CH1 or VH-CBeta were inserted into a linearized vector containing the human IgG4 / IgG1 CH2-CH3 constant region. The vector contains a CMV promoter and a human antibody heavy chain signal peptide. Plasmid connections, transformations, DNA preparations were performed using standard molecular biology protocols. Site-directed mutagenesis was conducted by PCR amplification using mutagenic primers and followed by DpnI digestion of the DNA template.

Protein Expression

[0492] The constructed heavy and light chain vectors were cotransfected into Expi293 cells (Thermofisher Scientific). The proportion of different vectors for cotransfection was adjusted according to the expected structure of the antibodies and the result of the initial expression was shown in SDS-PAGE. Briefly, 40 µg of plasmid and 108 µL of expifectamine were used to transfect a 40 ml volume of 1.2 x 108 cells. Intensifier 1 and Intensifier 2 were added 20 hours after transfection. The transfected cells were cultured at 37 ° C with 8% CO2 in an orbital shaker, rotating at 120 rpm. Five days after transfection, supernatants were collected by centrifugation and cell fragments were removed by filtration at 0.22 µm.

Expression detection by SDS-PAGE

[0493] The supernatant collected on day 5 was mixed with NuPAGE LDS Sample Buffer (4x), NuPAGE Sample Reducing Agent (10x) and H 2O. The reduced samples were heated to 75 ° C before loading into the gel. The gels were run using constant 200V for 35 minutes. Then, the gels were stained with SimplyBlue ™ SafeStain (Invitrogen, LC6065) and microwaved for 5 minutes. Discoloration was performed by incubation with water and micro-

waves for 7 minutes. The images of the gels were obtained using Universal HoodIII (Bio-Rad).

Purification Purification by chromatography on protein A

[0494] Protein resins MabSelect ™ SURE ™ (MSS) were obtained from GE Healthcare and packed in glass columns (BioRad). Purification by protein A chromatography was carried out at room temperature using a peristaltic pump as energy at a flow rate of 0.2 mL / min. After loading the samples, a volume of 10 columns of glycine at 100 mM, pH 3.5 was used for elution, and different fractions were collected. The protein concentration in different fractions was measured using a NanoDrop ™ 2000 (Thermo Fisher Scientific). The purity of the protein was detected by SDS-PAGE and SEC-HPLC.

Ion exchange chromatography (IEC)

[0495] IEC chromatographic experiments were performed using a 1 ml Hi trap SP HP column from GE Healthcare's life sciences with an ÄKTA Pure system (GE Healthcare). The method settings were programmed: wash the 10 CV column with wash buffer A (10 mM NaH2PO4, pH 6.0); apply the sample using the sample input; equilibrate the column with 10 CV wash buffer A (10 mM NaH2PO4, pH 6.0); elute the column with wash buffer A and wash buffer B (10 mM NaH2PO4, 1 M NaCl, pH 6.0). A gradient elution condition was applied as a coating step for 50 CV with 30% wash buffer B, a coating step for 5 CV with 100% wash buffer B and a fill step for 10 CV with 100% wash buffer wash buffer B. The fractions were collected as 0.5 mL per tube according to the UV absorbance value (the collection threshold was defined as 5 mAU).

Size exclusion chromatography (SEC)

[0496] Chromatographic experiments were performed using a Superdex ™ 10/300 GL 200 magnification column and a ÄKTA system from GE Healthcare.

The experiment was carried out using PBS (137 mM NaCl, 2.68 mM KCl, 1.76 mM KH2PO4, 10 mM NaH2PO4, pH 7.0) at 0.5 mL / min. The fractions were collected using the automated collection program (the collection value was defined as 5 mAU of UV absorbance) with 0.5 mL of each portion.

Ni Sepharose ™ Excel chromatography purification

[0497] Purification of 6xHis-tagged protein using excel Ni SepharoseTM resins Excel Chromatography Ni SepharoseTM purchased from GE Healthcare. The resin was packed in glass columns (BioRad). After the column was washed with 10 column volumes (CV) of ddH2O for the removal of the resin storage buffer, it was used for the purification of 6xHis-labeled proteins. Briefly, purification by Ni column was performed at room temperature using a peristaltic pump at a flow rate of 0.2 mL / min. After loading the sample, 10 CV of PBS (50 mM phosphate, 150 mM NaCl, pH 7.0) was used for washing, followed by 5 CV of elution buffer 1 (50 mM phosphate, 150 mM NaCl , 20 mM imidazole, pH 7.0) to remove loosely bound proteins. 10 CV of elution buffer 2 (50 mM phosphate, 150 mM NaCl, 500 mM imidazole, pH 7.0) was used to elute the bound protein.

After elution, the collected protein was measured using a NanoDrop ™ 2000 (Thermo Fisher Scientific). The purity of the eluted protein was detected by SDS-PAGE and SEC-HPLC. The column was regenerated using 10 CV of ddH2O, 10 CV of extraction buffer (50 mM of Tris, 500 mM of NaCl, 50 mM of EDTA, pH 7.4) for cleaning, 10 CV of guanidine hydrochloride a 6 M, pH 7.4 and 10 CV of 0.1 M nickel sulfate. The regenerated column was filled with 20% ethanol and stored at 4 ° C.

High performance liquid chromatography - Exclusion by size (SEC-HPLC)

[0498] The purity of the samples was analyzed using a TSK-GEL G3000SWXL column (7.8 mm × 300 mm) from Tosoh Bioscience and an Agilent 1200 HPLC system (Agilent Technologies). The column was equilibrated at a flow rate of 1.0 ml / min with phosphate buffer (50 mM sodium phosphate, 150 mM NaCl, pH 7.0).

After the 50 µL protein sample was filtered and injected, the UV absorbance at 280 nm was monitored. Purity was estimated by integrating the chromatograms.

Measurement of antibody concentration by ELISA

[0499] ELISA plates were coated with 200 ng / ml of goat anti-IgG-Fc (Fab) 2 form in the coating buffer (200 mM Na2CO3 / NaHCO3, pH 9.2). After overnight incubation at 4 ° C, the plates were washed once with PBS buffer using a deep well washer (Biotek ELx405). Then, the plates were blocked with 2% BSA in PBS buffer and incubated at room temperature for 1 hour. The plates were washed 3 times with wash buffer, and positive control antibody and diluted samples were added. After 2 hours of incubation, the plates were washed 6 times with 300 µL of washing buffer and biotinylated goat anti-human Ig-Fc (Bethyl, 100 µL / well, dilution 1: 5000 in 2% BSA) was added as detection antibody.

After the incubation and washing steps, SA-HRP (Invitrogen, dilution 1: 8000 in 2% BSA) was added. Then, the plates were incubated at room temperature for an additional 1 hour. The plates were washed 6 times with 300 µL of wash buffer / well. The TMB substrate was added and developed for 10 minutes. Stop solution (2 M HCl, 100 µL / well) was added to stop color development and absorbance was read at 450 nm using a plate reader (Molecular Device SpectraMax® M5e).

Target binding assays

[0500] The binding capacity of developed molecules was evaluated using the cell lines CD3 + Jurkat and CD19 + Ramos, respectively.

Both cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium (Invitrogen, Cat. No. 22400105), supplemented with 10% fetal bovine serum (SFB, Corning, Cat. No. 35-076 -CV).

[0501] Aliquots of 105 cells per well were collected and washed with 1% bovine serum albumin (BSA, BovoGen-BSAS), followed by incubation with studied antibodies diluted in series in a 96-well round bottom plate (Corning, Cat No. 3799) at 4 ° C for 1 hour. After washing twice with 1% BSA, the plates were further incubated with PE-conjugated goat anti-human IgG Fc antibodies (Jackson Immuno Research Laboratories, Cat. No. 109-115-098) at 4 ° C for 30 minutes. After the plates were washed twice again, the cells were analyzed by flow cytometry using a FACSCanto II cytometer (BDBiosciences) and the associated fluorescence intensity was quantified using the FlowJo software. Four-parameter nonlinear regression analysis was used to obtain the EC50 values in the Prism software (GraphPad Software, Inc).

Death of tumor cells driven by bispecific antibodies

[0502] To obtain human T cells, peripheral blood mononuclear cells (PBMCs) from healthy donors were then isolated by density centrifugation with Ficoll-Paque PLUS (GE Healthcare-17-1440-03) from heparinized venous blood . After grown in RPMI 1640 medium supplemented with 10% SFB, 1% penicillin / streptomycin solution (ScienCell, Cat. No. 0503), 50 units per ml of human IL-2 binding protein and 10 ng / ml of antibody OKT3 (EBioscience, Cat. No. 16-0037-85) for 6 days, PBMCs were passed through EasySep columns (Stemcell, Cat. No. 19053) for enrichment of CD8 + T cells. CD8 + T cells from the negative selection columns were used as effector cells.

[0503] In the cytotoxicity assay, CD19 + Raji cells, used as target cells, were pre-labeled with 20 nM CellTrace Far Red (Invitrogen, Cat.

No. C34564) at 37 ° C for 30 minutes. The cell pellets were then washed twice with phenol-free RPMI 1640 medium (Invitrogen, Cat. No. 11835030) supplemented with 10% SFB. In the 96-well round bottom plate (Corning, Cat. No. 3799), Far Red-labeled Raji B cells (20,000 cells / well) were incubated with isolated CD8 + T cells (ratio of target cells: effectors of 1 : 5) and bispecific antibodies diluted in series at 37 ° C for 4 hours. After incubation, 3 μM of propidium iodide (PI, Invitrogen, Cat. No. P3566) was added and mixed thoroughly to identify dead cells. After 15 minutes, the cells were analyzed by flow cytometry using a FACSCanto II cytometer. Bispecific antibody-mediated cytotoxicity can be defined as the percentage of PI-positive target cells in Far Red positive target cells. The EC 50 of cytotoxicity with T cell involvement was determined using the Prism software (GraphPad Software, Inc.).

Mass spectrometry characterization

[0504] The protein was diluted to 0.4 mg / mL and deglycosylated by incubation with 1μL of PNGase F (Glyko, GKE-5006D) (40: 1 protein / enzyme ratio) in 100 μL of 20 mM Tris buffer ( pH 8.0) at 37 ° C for at least 4 hours. An aliquot of deglycosylated bispecific antibodies was partially reduced by adding 2 μL of 1M DTT at a final concentration of 20 mM at room temperature for 15 minutes. Each sample at 2 µg was injected into a C4 Acquity UPLCBEH300 column (2.1 × 100 mm, 1.7 µm) at 0.4 ml / min. Mobile phase A consisted of 0.1% formic acid (FA) in HPLC grade water. Mobile phase B consisted of 0.1% AF in acetonitrile. For unreduced and reduced conditions, an efficient elution gradient of 24% B to 34% B was used from 3.0 to 15.0 minutes. After separation by UPLC RP, the bispecific protein mass in non-reducing and reducing conditions was detected by Waters Xevo G2 Q-TOF. The signs of MS were devolved using the BiophamaLynx 1.3 software. The theoretical average molecular weights of the mass of the light chain and the heavy chain were determined using the GPMaw program (v. 6.00).

DSF thermostability test

[0505] A DSF test was performed using the 7500 Fast real-time PCR system (Applied Biosystems). Briefly, 19 µL of the antibody solution was mixed with 1 µL of the SYPRO Orange 62.5x solution (Invitrogen) and added to a 96-well plate (Biosystems). The plate was heated from 26 ° C to 95 ° C at a rate of 2 ° C / min, and the resulting fluorescence data was collected.

The negative derivatives of the fluorescence changes in relation to different temperatures were calculated and the maximum value was defined as the Th melting temperature. If a protein has several split transitions, the first two Th were reported, called Th1 and Th2. Th1 is always interpreted as the formal melting temperature Tm to facilitate comparisons between different proteins. Data collection and Th calculation were performed automatically by the operating software. Since the graph of negative derivatives of the different temperatures was generated by the software, the point on the graph where the curve begins to decrease from a pre-transition baseline can be estimated approximately as the initial Ton temperature.

EXAMPLE 13: Identification of O-glycan

[0506] Previous mass spectrometry data found TCR-modified T3 light chain O-glycans. Unlike N-glycosylation sites, which can be located based on amino acid sequence patterns, O-glycosylation sites are difficult to predict from the sequence.

This T3 TCR-chimeric light chain was composed of the V region of the parental antibody T3, as well as the constant region of the TCR alpha chain. Both regions can potentially have O-glycosylation sites. The mass spectrometry analysis was conducted again on the original T3 monoclonal antibody and it was found that this parental antibody was free of O-glycans, which indicated that the O-glycans were located in the alpha constant region of the TCR.

[0507] O-glycosylation is known to occur mainly in the Ser or Thr residues, and there are 21 Ser / Thr residues in the sequence of the TCR alpha constant region (shown in bold in the sequence below). To locate the exact position of the O-glycosylation sites, Ala tracking was performed to replace each individual Ser / Thr with Ala, and 21 TCR-modified monospecific T3 molecules were constructed. The potential O-glycans in each mutant were released from the protein, labeled with 2-aminobenzoic acid and quantified by HPLC together with the Fluorescence Detector. The loss of the O-glycan signal can guide us to the location of the O-glycosylation site. 1 11 21 31 41

PDIQNPDPAV YQLRDSKSSD KSVCLFTDFD SQTQVSQSKD SDVYITDKCV 51 61 71 81 91

LDMRSMDFKS NSAVAWSQKS DFACANAFQN SIIPEDTFFP SPESS (SEQ ID NO: 411)

[0508] In order to identify and quantify the amount of O-glycan, a method based on acid hydrolysis and based on HPLC was developed. The sample was hydrolyzed with 2M TFA (trifluoroacetic acid) and the O-glycans monosaccharide was released. GalN released (galactosamine) from GalNAc (N-acetyl-D-galactosamine) from O-glycan and Gal (galactose) was stained with 2-aminobenzoic acid and analyzed by HPLC coupled with an FLD detector (Fluorescence Detector) and quantified by an external calibration curve. The content of GalN released was directly correlated with the amount of O-glycan, as it is the specific monosaccharide of O-glycans. The results reported that the amount of GalN per mole of protein, which means one mole of protein, contains the amount of mole of O-glycan.

[0509] Table 34 illustrates the quantified O-glycan levels in all mutants. The bispecific molecule E17-Design_2-QQQQ was used as a control protein. The data showed that there was 0.24 mol of O-glycans available in each mol of the E17-Design_2-QQQQ protein. As this is a bispecific antibody that has only one T3 light chain modified by TCR, the total O-glycan level of the two chains should be doubled, that is, around 0.48 mol / mol. Among all 21 mutants, most of them maintained the expected amount of O-glycan. Samples # 3, # 8, # 10 and # 20 had a slight decrease in signal.

Sample # 19 exhibited obvious loss of O-glycan. The signal was even lower than that of the control protein. Therefore, position S91 has been identified as the main O-glycosylation site. S19, S36, S41 and S94 were identified as possible Oglycosylation sites.

TABLE 34. O-glycans quantified in several unique Ala mutants (residue numbering was listed in Figure 19A) NO. of Drawing WBP3438 NO. Analytical AS1803474 Analysis of Test Item SOP NO. PD-PAS-LAB-090-02 Monosaccharide No. of Test Result (mol / mol protein) Sample ID Sample Galactosamine 196388 E17-Design_2-QQQQ 0.24 (x2) 1 T3.uIgG4.SP (S16A) 0, 41 2 T3.uIgG4.SP (S18A) 0.43 3 T3.uIgG4.SP (S19A) 0.38 4 T3.uIgG4.SP (S22A) 0.45 5 T3.uIgG4.SP (T27A) 0.57 6 T3.uIgG4.SP (S31A) 0.44 7 T3.uIgG4.SP (T33A) 0.42 8 T3.uIgG4.SP (S36A) 0.36 9 T3.uIgG4.SP (S38A) 0.47 10 T3. uIgG4.SP (S41A) 0.37 11 T3.uIgG4.SP (T46A) 0.50 12 T3.uIgG4.SP (S55A) 0.43 13 T3.uIgG4.SP (S60A) 0.53 14 T3.uIgG4. SP (S62A) 0.56 15 T3.uIgG4.SP (S67A) 0.57 16 T3.uIgG4.SP (S70A) 0.52 17 T3.uIgG4.SP (S81A) 0.53 18 T3.uIgG4.SP ( T87A) 0.55 19 T3.uIgG4.SP (S91A) 0.12

AT THE. of Drawing WBP3438 NO. Analytical AS1803474 Analysis of Test Item SOP NO. PD-PAS-LAB-090-02 Monosaccharide No. of Test Result (mol / mol protein) Sample ID Sample Galactosamine 20 T3.uIgG4.SP (S94A) 0.32 21 T3.uIgG4.SP (S95A) 0, 61 EXAMPLE 14: Binding to the Fcγ, C1q and FcRn receptor Methods Affinity binding to the Fcγ receptor by SPR

[0510] The antibody binding affinity to FcγRs was detected using Biacore T200 (or Biacore 8K). Each receiver was captured on a CM5 (GE) sensor chip immobilized with anti-his antibody. The antibodies in different concentrations were injected on the sensor chip at a flow rate of 30 µL / min for a 60 s association phase, followed by 60 s dissociation. The chip was then regenerated by 10 mM glycine (pH 1.5) after each binding cycle.

[0511] The sensograms of the buffer channel and reference surface (blank) were subtracted from the test sensograms. The experimental data were adjusted by the 1: 1 model using Langmiur analysis (for FcγRI) or steady state model (for other receptors). The molecular weight of 150 KDa was used to calculate the molar concentration of the antibodies.

Binding to C1q by ELISA

[0512] ELISA plates (Nunc) were coated with antibody samples at 3μg / ml overnight at 4 ° C. After blocking and washing, the C1q was gradually diluted from 600 μg / mL and incubated at room temperature for 2 hours. The plates were then washed and subsequently incubated with sheep anti-human C1q-HRP Ab for 1 hour. After washing, the TMB substrate was added and the interaction was interrupted by 2M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

FcRn binding affinity for SPR

[0513] The antibody binding affinity to FcRn was detected using Biacore T200 (or Biacore 8K). Each antibody was immobilized on the CM5 (GE) sensor chip. The FcRn in different concentrations in the running buffer (50 mM Na2HPO4 / NaH2PO4, 150 mM NaCl, 0.05% Tween20, pH 6.0) was injected onto the sensor chip at a flow rate of 30 µL / min for a 60 s association phase, followed by 60 s decoupling. The chip was then regenerated by PBS 1X (pH 7.4) after each binding cycle.

[0514] The sensograms of the buffer channel and reference surface were subtracted from the test sensograms. The experimental data were adjusted using the steady state model. A molecular weight of 45 KDa was used to calculate the molar concentration of FcRn.

Results

[0515] Since all of the IgG1s mentioned above were IgG1 with LALA mutation, the binding activity of E17-Design_2-QQQQ in both IgG4 and wild type IgG1 (T3U4.E17-2. (2) .uIgG1 (wild type IgG1 with knob-into-holes)) FcγRI, FcγRIIa (H167), FcγRIIa (R167), FcγRIIb, FcγRIIIa (F176), FcγRIIIa (V176) and FcγRIIIb were investigated by SPR.

[0516] The relevant sequences of construction T3U4.E17-2. (2) .uIgG1 are provided below.

LC-NLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWSSYPYTFGQGTK DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAPKLLIYSTS U4 (SEQ ID LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL NO: 371) QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

TKSFNRGEC QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQRLEWIGYI DPYNGDTTYNQKFKGRVTITRDMSTSTAYMELSSLRSEDTAVYYCLTTAYA

MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV T3U4.E17- TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK U4-HC

2. (2) .uIgG1 PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT (SEQ ID NO: 372) PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVSV

VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEM TKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSK

LC-T3 LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPK (LLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCTQSHTLRT SEQ ID NO: 373) FGGGTKVEIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDS

DVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPSP ESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLEWMGWI SPGNVNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYS LYYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATG

FYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATF T3-HC

WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRASDKTHTCP (SEQ ID PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY NO: 374) VDGVEVQYKNY

APIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

[0517] Affinities are summarized in Table 35 (IgG4) and Table 36 (wild type IgG1). Both molecules showed a typical affinity for binding to human IgG4 and wild-type IgG1 to all Fcγ receptors.

TABLE 35. Affinity of IgG4 to Fc receptor by SPR Receptor Fc KD (M) FcγRI 9,79E-09 FcγRIIa (H167) 2,05E-05 FcγRIIa (R167) 1,58E-05 FcγRIIb 2,41E-05 FcγRIIIa (F176 ) 2.93E-05 FcγRIIIa (V176) 1.40E-05 FcγRIIIb> 4.10E-05 TABLE 36, Affinity of wild-type IgG1 to the Fc receptor by SPR Receiver Fc KD (M) FcγRI 1.30E-09 FcγRIIa ( H167) 3,58E-06 FcγRIIa (R167) 4.83E-06 FcγRIIb 8.07E-06 FcγRIIIa (F176) 2,08E-06 FcγRIIIa (V176) 6.44E-07 FcγRIIIb 5.16E-06

[0518] The antibody binding activity to C1Q was tested by ELISA (Figures 21A-21B). E17-Design_2-QQQQ in IgG4 showed no sign of binding in the ELISA (Figure 21A), while E17-Design_2-QQQQ in wild-type IgG1 and the control human IgG1 antibody showed normal binding signal (Figure 21B).

EXAMPLE 15: Symmetrical formats G19, G19R, G25, G25R

[0519] Therapeutic antibody targets, such as CD3 x CD19, benefit from a bispecific antibody with binding to monovalent CD3 for safety reasons. With this in mind, the asymmetric bispecific formats E17 and F16 were successfully developed and generated, through the integration of WuXiBody Fab, as well as the “knob-into-holes” techniques. Some bispecific targets such as CTLA-4 x PD-1, however, benefit from a symmetrical shape, which can mount two different antibodies while maintaining their original valences (ie, tetravalent in total) to achieve the desired synergistic effects. The main unit of WuXiBody is a chimeric Fab, which can be easily incorporated in asymmetric and symmetrical formats to ensure the correct pairing of cognate light-heavy chains. The four symmetrical formats based on WuXiBody, called G19, G19R, G25 and G25R, were developed.

[0520] Figure 22 provides a schematic description of four symmetrical formats. At G19 and G25, two chimeric WuXiBody Fabs were grafted to the C-terminal and N-terminal end of a normal antibody, respectively. The difference between G19 and G19R, or between G25 and G25R, is the inverted locations of the normal and chimeric Fab in each individual format. The heavy parts of two Fabs, as well as the IgG-Fc, were integrated into a chain, while the two light chains were free to fold and assemble independently. When three vectors were cotransfected into host cells, the heavy-heavy association was expected to occur as normal antibodies during the expression process, while each light chain was expected to self-assemble into its own cognate partner in the heavy chain.

[0521] Bisexual CTLA-4 x PD-1 antibodies in symmetrical WuXiBody format have been developed. A new anti-PD-1 antibody W3055_1.153.7 (named U6) and a commercial anti-CTLA-4 ipilimumab antibody (named T1) were adopted to integrate the new formats. The IgG4 isotype was chosen to guarantee the depletion of the ADCC and CDC effect in the molecule. As U6 and T1 could be placed on the upper or lower side of the format (named U6T1 and T1U6, respectively), the unique G19 format was used primarily to investigate the two cases.

[0522] Relevant WuXiBody sequences tested are provided below: No. of Samples Sequences Plasmid

LC-YGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLI T1 (SEQ ID NO: 375 FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV) QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVT FISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCAR TGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP

KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF T1-U6 HC-NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE T1U6.G19.I PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT (SEQ ID NO: 376 PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL GG4)

SLGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASG FTFSSHAMSWVRQAPGKGLEWVSTITGGGGSIYYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCAKNRAGEGYFDYWGQGTLVTVLEDLKNV FPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSG VCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLS ENDEWTQDRAKPVTQIVSAEAWGR

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 377 GTKLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSD) VYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPS

PESS

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 378 GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW) KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTH

EGSTVEKTVAPTECS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

NRAGEGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL U6T1.G19.I VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG GG4 TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK U6-T1-PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ HC (SEQ ID NO: 379 FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR) EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGGGGGSGGGGSGGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDN SKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVLEDLKNV FPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSG

No. of Samples Sequences Plasmid

VCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLS ENDEWTQDRAKPVTQIVSAEAWGR

LC-YGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLI T1 (SEQ ID NO: 380 FGQGTKVEIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSK) DSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTF

FPSPESS

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 381 GTKLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSD) VYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPS

PESS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK NRAGEGYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSS RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG

RYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE U6-T1-HC DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE U6T1.G19R. YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC (SEQ ID NO: IgG4 LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR 382)

WQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGG SQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWV TFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCA RTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRV

LC-YGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLI T1 (SEQ ID NO: 383 FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV) QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 384 GTKLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSD) VYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPS

PESS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

NRAGEGYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL U6T1.G25.I

VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSS gG4

RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG U6-T1-HC RGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVR (SEQ ID NO: QAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLR 385) AEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRST

SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK

No. of Samples Sequences Plasmid

TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK

LC-YGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLI T1 (SEQ ID NO: 386 FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV) QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 387 GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW) KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTH

EGSTVEKTVAPTECS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK NRAGEGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVGGGGSGGGGSQVQLVESGGGVVQPGRSL

RLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGR U6-T1-HC FTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTV U6T1.G25R. LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVN (SEQ ID NO: IgG4 GKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQ 388)

VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPEFLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK

LC-YGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLI T1 (SEQ ID NO: 389 FGQGTKVEIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSK) DSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTF

FPSPESS

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 390 GTKLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSD) VYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPS

PESS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS

TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK U6T4.G26.I

NRAGEGYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL gG4 VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSS U6-HC-T4-LC

RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG (SEQ ID NO: RGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCRSSQSLLNSDGNTYL 391) YWYLQKPGQSPQLL

VGVYYCVQGTHDPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

No. of Samples Sequences Plasmid

QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIRKPPGKGMEWI GYISYSGNTNYNPSLKSRVTISRDTSKNQFFLKLSSVTAADTAVYYCAS MMVPHYYVMDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC

LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL T4 GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP HC (SEQ ID NO: 392 KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE) QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP

REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 393 GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW) KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTH

EGSTVEKTVAPTECS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK NRAGEGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK

PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ-T5-U6 HC FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR U6T5.G19.I EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT (SEQ ID NO: 394 TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS GG4)

LSLGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKAS GYTFTNYFMNWVRQAPGQGLEWMGRVDPEQGRADYAEKFKKRVTITADK STSTAYMELSSLRSEDTAVYYCARRAMDNYGFAYWGQGTLVTVLEDLKN VFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHS GVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGL SENDEWTQDRAKPVTQIVSAEAWGR

LC-TSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFG EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHG T5 (SEQ ID NO: 395 GGTKVEIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDS) DVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFP

SPESS

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 396 GTKLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSD) VYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPS

PESS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK NRAGEGYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL

VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSS U6T5.G19R. RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG RYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE IgG4-T5-U6 DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE HC (SEQ ID NO: 397 YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC) LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR

WQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSGGGG SQVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVRQAPGQGLEWM GRVDPEQGRADYAEKFKKRVTITADKSTSTAYMELSSLRSEDTAVYYCA RRAMDNYGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRV

No. of Samples Sequences Plasmid

LC-TSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFG EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHG T5 (SEQ ID NO: 398 GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW) KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 399 GTKLTVLPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSD) VYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPS

PESS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK NRAGEGYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSS RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG

RGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVR-T5-U6 HC QAPGQGLEWMGRVDPEQGRADYAEKFKKRVTITADKSTSTAYMELSSLR U6T5.G25.I SEDTAVYYCARRAMDNYGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRS (SEQ ID NO: 400 TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS GG4)

SVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK

LC-TSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFG EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHG T5 (SEQ ID NO: 401 GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW) KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

LC-DSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYYCQVWDSIWVFGG SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAPVLVIYR U6 (SEQ ID NO: 402 GTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW) KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTH

EGSTVEKTVAPTECS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK NRAGEGYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVGGGGSGGGGSQVQLVQSGAEVKKPGSSV

KVSCKASGYTFTNYFMNWVRQAPGQGLEWMGRVDPEQGRADYAEKFKKR U6-T5-HC VTITADKSTSTAYMELSSLRSEDTAVYYCARRAMDNYGFAYWGQGTLVT U6T5.G25R. VLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV (SEQ ID NO: IgG4 NGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRC 403)

QVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK

LC-TSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHHWSNTQWTFG EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSPKLWVHG T5 (SEQ ID NO: 404 GGTKVEIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDS) DVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFP

SPESS

No. of Samples Sequences Plasmid

QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIRKPPGKGMEWI GYISYSGNTNYNPSLKSRVTISRDTSKNQFFLKLSSVTAADTAVYYCAS MMVPHYYVMDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP

KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE T4-HC-U6-LC

QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP (SEQ ID NO: REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK 405) TTPPVLDSDGS

SLSLGKGGGGSGGGGSGGGGSGGGGSSYELTQPLSVSVALGQTARITCG GDNIGNKDVHWYQQKPGQAPVLVIYRDSNRPSGIPEGFSGSNSGNTATL

TISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPDIQNPDPAVYQLRDS T4U6.G27.I KSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA gG4 WSQKSDFEDTFPSIPS

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS

TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK U6 HC-NRAGEGYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL (SEQ ID NO: 406 VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSS) RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG

R

LC-T4 DIVMTQTPLSLSVTPGQPASISCRSSQSLLNSDGNTYLYWYLQKPGQSP QLLIYLVSKLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTH (SEQ ID NO: 407 DPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR) EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHQGLSSPVTKSFNRGEC QVQLQESGPGLVKPSETLSLTCSVTYHTITSGYDWTWIRKPPGKGMEWI GYISYSGNTNYNPSLKSRVTISRDTSKNQFFLKLSSVTAADTAVYYCAS

MMVPHYYVMDAWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC T4-HC-LC-LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL U6 (SEQ ID NO: 408 GTKTYTCNVDHKPSNTKVDKRVGGGGSGGGGSSYELTQPLSVSVALGQT) ARITCGGDNIGNKDVHWYQQKPGQAPVLVIYRDSNRPSGIPEGFSGSNS

GNTATLTISRAQAGDEADYYCQVWDSIWVFGGGTKLTVLPDIQNPDPAV YQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFK SNSAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVS

TITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK T4U6.G26R. NRAGEGYFDYWGQGTLVTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL IgG4 HC-RLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWG VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSS U6 (SEQ ID NO: 409 RYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE) DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE

YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSLG

LC-T4 DIVMTQTPLSLSVTPGQPASISCRSSQSLLNSDGNTYLYWYLQKPGQSP QLLIYLVSKLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGTH (SEQ ID NO: 410 DPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR) EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHQGLSSPVTKSFNRGEC

[0523] Both U6T1 and T1U6 constructs were expressed normally in the Expi293 system, and the expressed protein reached about 90% purity after purification in one step of protein A chromatography. Figures 23A-23B showed the characterizations by SDS- PAGE and SEC-HPLC of the purified proteins.

To inspect its binding capacity, cell-based binding assays for the PD-1 and CTLA-4 targets were subsequently conducted. Figures 24A-24B showed that both U6 and T1 had reduced binding, if located on the underside of the shape. Considering that the function of PD-1 is of relatively greater importance than that of CTLA-4 (antibodies against CTLA-4 are known to have a more serious side effect), the PD-1 binding side was placed on the upper side to maximize the connection to U6 (ie U6T1 instead of T1U6), and to test how to optimize the connection to CTLA-4 located on the bottom side.

[0524] The other three formats of WuXiBody G19R, G25 and G25R (shown in Figure 22) were investigated as well. In addition, a reference antibody AK-104 (Akeso Biopharma, Inc.), which is a bispecific PD-1 / CTLA-4 antibody used in the clinical trial, was obtained and used as a control for direct comparison.

[0525] Due to the importance of the PD-1 function, U6 was maintained on the upper side of all formats to maximize binding to PD-1, while T1 was maintained on the lower side to perform proper binding to CTLA-4. All constructed molecules were well expressed in Expi293 and easily reached> 90% purity after purification in one step of protein A chromatography. Figures 25A-25B showed the purified proteins characterized by SDS-PAGE, as well as SEC-HPLC.

[0526] Cell-based PD-1 and CTLA-4 binding assays were then performed to verify the binding capacity of all newly constructed molecules. Figures 26A-26B showed comparisons of the binding curves between the constructs developed and the reference antibody. The data showed that all proteins had a PD-1 binding very similar to the reference antibody. In addition, binding to CTLA-4 improved significantly in the G25 and G25R format and achieved performance comparable to the reference antibody (<=

2 times). The G19R format, however, has not yet worked well. It is likely that G19 and G19R shared the same problem that prevented T1 from actually binding.

[0527] The functions of the molecules were further characterized by the inspection of their competition capabilities for each ligand of two targets, PD-L1 and CD80.

Figures 27A-27B confirmed that these molecules have a performance comparable to the reference value to compete with PD-L1. For the CTLA-4 side, the G25R format showed a capacity similar to that of the reference when competing with the CD80. The other two formats had relatively worse results. The difference between G25 and G25R is the location of the region in the TCR. It seems that converting T1 to WuXiBody format facilitated T1's activity, although T1 was still below U6. This provided a good example of demonstration that functional leaders could be effectively scanned against the limited number of formats derived from WuXiBody.

[0528] Accordingly, a functional bispecific PD-1 / CTLA-4 antibody similar to the reference antibody was obtained. WuXiBody formats are very universal, that is, any new antibody can fit into these formats and perform its function. If a good parental antibody is available, it can be used to create a molecule superior to the reference antibody.

[0529] To prove the concept, another anti-CTLA-4 antibody W3162_1.154.8-z35 (called T5), which has a much stronger affinity than Ipilimumab, was developed and implemented in all four formats G19, G19R, G25 and G25R shown in Figure 22. Again, all new constructs were well expressed in Expi293 cells and easily purified by one-step protein A chromatography. The degrees of purity of the proteins were shown in Figures 28A-28B.

[0530] The connections of all U6T5 molecules, the previously identified U6T1.G25R molecule, as well as the reference antibody were all conducted and compared. The results were listed in Figures 29A-29B. The PD-1 side maintained the original binding behaviors as noted earlier, because no PD-1 antibodies were substituted in either format. However, for the CTLA-4 side, all of the U6T5 constructs (even the G19 and G19R formats) exhibited obvious superior connections over the U6T1.G25R, as well as to the reference molecule. U6T5.G25 was the strongest among all the new proteins, which had an EC50 improved by 1.6x and higher values> 3x compared to the reference antibody. This molecule was further characterized in the double bond ELISA assay and in the FACS competition assays. Figure 30 proved the effective double bonds of the molecules to both targets simultaneously. The data in Figures 31A-31B confirmed that U6T5.G25 had significantly better competition capacity with the CD80 for CTLA-4.

This proved that WuXiBody formats were flexible enough to handle different parental antibodies. The upper part of a parental antibody can be well preserved and reflected when the molecule is connected to WuXiBody shapes.

[0531] The thermal stability of the molecules that comprised all four symmetrical shapes was characterized. Most of the molecules showed the melting temperature around 60 ° C (shown in Table 37), which is consistent with the asymmetric shape shown above.

TABLE 37: Representative antibody temperatures in WuXiBody formats Th1 Th2 concentration Protein name Isotype pI Buffer (mg / ml) (℃) (℃) T1U6.G19.IgG4 IgG4, kappa, lambda 5.92 PBS 1.3 60, 8 69.9 U6T1.G19.IgG4 IgG4, kappa, lambda 5.92 PBS 0.9 59.1 72.8 U6T1.G25R.IgG4 IgG4, kappa, lambda 6.06 PBS 1.385 60.8 73.9

U6T5.G19.IgG4 IgG4, kappa, lambda 5.93 PBS 0.6 56.2 74.1 U6T5.G19R.IgG4 IgG4, kappa, lambda 5.87 PBS 1.2 63.4 - U6T5.G25.IgG4 IgG4 , kappa, lambda 5.93 PBS 0.7 63.4 - U6T5.G25R.uIgG4 IgG4, kappa, lambda 5.99 PBS 0.5 57.2 74.1 EXAMPLE 16: Chimeric Fab with light-heavy exchange

[0532] In total, 111 potential formats based on WuXiBody have been successfully developed. In addition to the E17, F16, G19, G19R, G25 and G25R shown above, some formats were also developed with chimeric Fab with TCR with light-heavy cross. These were named G26, G26R and G27, shown in Figure 32. This time, the antibody pair U6 and T4 was used, where T4 was an anti-CTLA-4 antibody, WBP3162-1.146.19-z12. The T4U6 pair was developed in the G27 and G26R format. Figures 33A-33B showed the purified protein characterized by SDS-PAGE and SEC-HPLC. Although both proteins were expressed, T4U6.G27.IgG4 had a low degree of purity, but T4U6.G26R.IgG4 had correct molecular weight and high purity. The binding capacity of the last molecule was characterized in FACS binding. Figures 34A-34B showed that the binding to PD-1, once located on the lower side, was affected, while the binding side of the CTLA showed complete recovery, as it was placed on the upper side of the shape.

[0533] The U6T4 pair was tested in the G26 format. Both the expression and purification steps worked well, as shown in Figures 35A-35B.

The connections evidenced by ELISA and FACS were conducted and the data were presented in Figures 36A-36D. These data proved that the chimeric Fab with light-heavy crossover can still work well. The melting temperature of this molecule is about 63.4, as shown in Table 38.

TABLE 38. Melting temperature of U6T4.G26.IgG4 Concentration Th1 Th 2 Protein name Isotype pI Buffer (mg / ml) (℃) (℃) U6T4.G26.IgG4 IgG4, kappa, lambda 5.93 PBS 0.8 63.4 - EXAMPLE 17: Anti-CD13 x CD19 WuXiBody Bispecific

Basics Target Biology

[0534] Human CD19 is a type I transmembrane protein belonging to the immunoglobulin superfamily (Carter et al., Curr Dir Autoimmun, 2004, 7: 4-32). It is expressed in most B cells, but is not detected in plasma cells, stem cells or in the normal myeloid lineage (Tedder, Nat Rev Rheumatol, 2009, 5 (10): 572-577). CD19 is critically involved in the establishment of intrinsic B cell signaling thresholds through the modulation of B cell receptor dependent (BCR) independent signaling (Wang et al., Experimental Hematology & Oncology, 2012, 1:36). CD19 has a broader expression than CD20. The expression pattern of CD19 is maintained in B cell neoplasms, covering all subtypes of B cell lymphoma, from indolent to aggressive forms, as well as chronic B cell lymphocytic leukemia and acute non-T lymphoblastic leukemia and allows targeting tumor indications of early B cells, such as acute lymphoblastic leukemia (ALL), which cannot be affected by Rituximab. Various monoclonal CD19 antibodies have been explored for lymphoma therapy (U.S. Patent Application Publication No. 20140072587 A1, U.S. Patent No. 8,242,252 B2, and U.S. Patent No. 8,097,703 B2).

[0535] The CD3 T cell coreceptor is a protein complex composed of four distinct chains, a CD3Gama chain, a CD3delta chain and two CD3epsilon chains. The four chains associate with a molecule known as the T cell receptor (TCR) and the zeta chain to generate an activation signal in T lymphocytes. The TCRs, the zeta chain, and the CD3 molecules make up the TCR complex, in which the TCR, as a subunit, recognizes and binds to the antigen, and CD3, as a subunit, transfers and transports antigenic stimulation to the signaling pathway, and ultimately regulates T cell activity. CD3 protein is present in virtually all T cells. The CD3-TCR complex modulates cell functions

T, both in innate and adaptive immune responses, as well as cellular and humoral immune functions. These include the elimination of pathogenic organisms and the control of tumor growth by a wide variety of cytotoxic effects.

Mouse monoclonal antibodies specific for human CD3, such as OKT3 (Kung et al., Science, 1979, 206: 347-9), were the first generation CD3 antibodies developed for treatment. Although OKT3 has a strong immunosuppressive power, its clinical use has been hampered due to serious side effects linked to its immunogenic and mitogenic potentials (Chatenoud, Nature Reviews, 2003, 3: 123-132). OKT3 induced an antiglobulin response, promoting its own rapid clearance and neutralization (Chatenoud et al., Eur. J. Immunol., 1982, 137: 830-8). In addition, OKT3 induced T cell proliferation and cytokine production in vitro, and led to a large-scale release of cytokines in vivo (Hirsch et al., J. Immunol, 1989, 142: 737-43). Such serious side effects limited the more widespread use of OKT3 in transplantation, as well as the extent of its use in other clinical fields, such as autoimmunity (Id.).

[0536] A bispecific antibody targeting CD3 and CD19 can bind T cells and B cells simultaneously. Since the bispecific antibody binds to a CD3 positive T cell and a CD19 positive B cell, a cytolytic synapse is formed. Cytotoxicity is then induced by the release of perforin and granzymes from granules in the cytotoxic T cell, the latter inducing apoptosis and lysis of the malignant B cell.

[0537] Blinatumomab activity has been proven to be independent of antigen presentation by MHC class I and TCR recognition. Therefore, it can bypass a variety of tumor-mediated immune escape mechanisms, such as impairment of antigen presenting machines and activation of negative co-stimulating signals in the tumor microenvironment.

Unmet medical needs

[0538] The treatment of acute lymphoblastic leukemia (ALL) in adults remains challenging and new therapies are needed. With current therapies, response rates vary from 30 to 50%, depending on the duration of the initial remission, age and cytogenetics. The overall response rates for a subset of non-Hodgkin's lymphoma (NHL) are now greater than 90% in regimens employing the first generation of anti-CD20 antibodies. However, several NHL subtypes are not as responsive to these therapies, and most patients with responsive NHL end up relapsing after the combined standard immunotherapy / chemotherapy regimen. Thus, new first-line therapies and new rescue schemes are needed for these unmet needs.

Materials and methods Generation of cell line of cynomolgus monkey that expresses CD19

[0539] The complete human or cynomolgus monkey CD19 gene was cloned into the pcDNA3.3 vector. Each expression vector was then transfected into CHO-K1 cells, respectively, using Lipofectamine 2000. The cells were cultured in F12-K with 10% SFB. Blasticidin was added 24-48 hours after transfection. After two to three passages of the selection, the cells were enriched with anti-CD19 antibody conjugated to PE and anti-PE microspheres (Miltenyi - 013- 048-801). The stable single cell clones were isolated by limiting dilution and screened by FACS using anti-CD19 antibody.

Tumor strains that express target

[0540] The Raji and Jurkat cells came from the ATCC. The Ramos cell came from the ECACC. All tumor cells were cultured in RPMI1640 / 10% SFB.

Construction of the WuXiBody W3438-T3U4.E17-1.uIgG4.SP and W3438- T3U4.F16-1.uIgG4.SP

[0541] The VL, VH, Ck, CH1 genes were amplified by PCR from existing internal DNA models. The CAlfa and CBeta genes were synthesized by Genewiz Inc. The anti CD3 chimeric or anti CD19 native light chain genes were inserted into a linearized vector containing a CMV promoter and a kappa signal peptide. The DNA fragments of Anti CD3 VH-CBeta were inserted into a linearized vector containing the human IgG4S228P CH2-CH3 constant region with a knob mutation. The DNA fragments of Anti CD 19 VH-CH1 were inserted into a linearized vector containing the CH2-CH3 constant region of human IgG4S228P with a hole mutation. The vector contains a CMV promoter and a human antibody heavy chain signal peptide.

Expression and purification of W3438-T3U4.E17-1.uIgG4.SP and W3438- T3U4.F16-1.uIgG4.SP

[0542] The heavy and light chain expression plasmids were cotransfected to Expi293 cells using the Expi293 expression system kit (ThermoFisher-A14635) according to the manufacturer's instructions. Five days after transfection, supernatants were collected and the protein was purified using protein A column (GE Healthcare-17543802) and additional column for size exclusion (GE Healthcare-17104301). The concentration of antibodies was measured by Nano Drop. Protein purity was assessed by SDS-PAGE and HPLC-SEC.

Target connection by FACS

[0543] The binding of bispecific antibodies to cells that express CD3 and CD19 was assessed using Jurkat and Ramos, respectively. A non-relevant antibody was used as an isotype control. The cells were spread in 96-well plates (Corning-3799) at a density of 105 cells / well and washed with PBS / 1% BSA. The antibodies were serially diluted and incubated with the cells at 4 ° C for 1 hour. PE-conjugated goat anti-human IgG Fc antibody

(Jackson-109-115-098) was used for detection. After washing and resuspension, the cells were analyzed by flow cytometry (Canto II, BD Biosciences). The data were analyzed using the FlowJo software. Four-parameter nonlinear regression analysis was used to calculate EC50 values using the GraphPad Prism software.

Connection to cinomolgo CD3

[0544] The binding of the bispecific CD3 × CD19 antibody to cinomolgo CD3 was tested by protein binding ELISA. 96 well ELISA plates of high protein binding (Nunc MaxiSorp, ThermoFisher, Thermo-442404) were coated overnight at 4 ° C with 100 µL of 1 μg / mL of cinomolgo CD3 epsilon protein (Acro, # CDE- C5226) in carbonate-bicarbonate buffer (20 mM Na 2CO3, 180 mM NaHCO3, pH 9.2). All wells were washed once with 300 μL of PBS / 0.5% Tween-20 (v / v) per well. The wells were then blocked for one hour at room temperature with 200 μL of PBS / 2% BSA (BOVOGEN, #BSAS) per well and washed three times with 300 μL per well of PBS / 0.5% Tween-20 ( v / v) For the binding of the primary antibody, the bispecific CD3 × CD19 antibody serially diluted in PBS / 2% BSA was transferred to the relevant wells and incubated at room temperature for two hours. The plates were washed three times in the same manner as before adding 100 µL of secondary antibody of 100 ng / mL of anti-human IgG Fc-goat HRP (Bethyl, # A80-304P). The plates were incubated at room temperature for one hour, followed by six washes as described above. For the detection of binding, 100 µL of tetramethylbenzidine (TMB) substrate solution (Sigma-860336) was added to all wells for 10 minutes at room temperature in the dark before stopping the reaction with 100 µL of 2M HCl. The extent of binding of the bispecific antibody to the cinomolg CD3 was determined by measuring the absorbance at OD450 using the SpectraMax® M5e microplate reader. Whenever appropriate, binding EC 50 values were obtained by non-linear regression analysis of four parameters, using the GraphPad Prism 5 software.

Connection to cinomolgo CD19

[0545] The binding of the bispecific CD3 × CD19 antibody to the cynomolg CD19 target protein expressed in CHOK1 cells was determined by flow cytometry analysis. Briefly, the stable cell line that overexpresses the cinomolg CD19 (WBP701.CHOK1.cPro1.C9, WuXiBiologics) was collected with trypsin and diluted to 1 x 106 cells / mL in 1% BSA / PBS 1X. 1 x 105 cells / well (100 µL) was added to each well of a 96-well U-bottom plate (Corning, # 3799) and centrifuged at 1500 rpm (Eppendorf, # 5810R) for 5 minutes before removing the supernatant. Antibodies serially diluted in 1% BSA / PBS 1X were added at 100 µL / well to the pelleted cells and incubated at 4 ° C for 1 hour.

An unrelated hIgG4 antibody was used as an isotype control. The cells were washed twice with 180 µL / well of 1% BSA / PBS 1X by centrifugation at 1500 rpm for 5 minutes at 4 ° C. The pelleted cells were resuspended in 100 µL / well of fluorescently labeled anti-human IgG Fc antibody (Jackson, # 109-115-098) diluted 1: 150 in 1% BSA / PBS 1X for 30 minutes at 4 ° C in the dark. The cells were then washed twice as described above. After the final wash, the cells were resuspended in 80 µL of 1% BSA / PBS 1X and fluorescence values were measured with a FACS Canto II cytometer (BDBiosciences). The amount of bispecific anti-CD19 and CD3 antibody bound to the cell surface was assessed by measuring mean fluorescence (MFI). The raw data from FACS were analyzed using the FlowJo software; wells containing no antibody or secondary antibody were used only to establish background fluorescence. The binding EC50 values were obtained by non-linear regression analysis of four parameters using the GraphPad Prism 5 software.

Affinity for FACS

[0546] The binding affinity for CD3 and CD19 was determined by flow cytometry using Jurkat and Ramos cells, respectively. The cells were transferred to 96-well U-bottom (BD) plates at a density of 5x10 4 cells / well. The antibodies to be tested were serially diluted 1: 2 times in 1% PBS 1X / 1% BSA and incubated with the cells at 4 ° C for 1 hour. Then, the plates were centrifuged at 1500 rpm for 4 minutes and the supernatant discarded. The secondary antibody, goat anti-human IgG Fc conjugated to Alexa 647 (Jackson, Cat # 109-605-098) or goat anti-His FITC conjugated (Bethyl, Cat # A190-113F) was added to the resuspended cells and incubated at 4 ° C in the dark for 30 min. The cells were washed once and resuspended in 100 mL of 1X PBS / 1% BSA. The fluorescence intensity was measured by flow cytometry (BD Canto II) and analyzed by FlowJo.

The fluorescence intensity was converted into linked molecules / cells based on quantitative spheres (QuantumTM MESF Kits, Bangs Laboratories). K D was calculated by Graphpad Prism 5.

Double binding to target cells

[0547] The ability of bispecific antibodies to bind CD3 T cells and CD19 B cells has been tested by FACS. Jurkat and Raji cells were pre-labeled separately with 20 nM CellTrace Far Red (Invitrogen-C34564) and 50 nM calcein-AM (Invitrogen-C3099) at 37 ° C for 30 min, at a density of 1 x 10 6 cells / ml. The pelleted pre-labeled cells were washed twice with PBS / 1% BSA, then mixed 1: 1 to a final density of 1 x 10 6 cells / ml. The cell mixture was centrifuged and resuspended with 10 nM of antibody followed by 1 hour of incubation. The cell mixture was analyzed by flow cytometry immediately after incubation. The bridging percentage was calculated as the percentage of events marked simultaneously with Far-Red and calcein.

Cytotoxicity assay

[0548] Fresh peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation by Ficoll-Paque PLUS (GE Healthcare-17-1440-03) from heparinized venous blood. The PBMCs obtained were passed through EasySep columns (Stemcell-19053) to enrich CD8 + T cells, which were used as effector cells. The effectiveness of antibodies to mediate tumor cell lysis by CD8 + T cells was determined by flow cytometry. In the cytotoxicity assay, Raji CD19 B cells as target cells were pre-labeled with 20 nM CellTrace Far Red (Invitrogen-C34564) at 37 ° C for 30 min, followed by washing the cell pellets twice with RPMI 1640 without phenol (Invitrogen-11835030) supplemented with 1% SFB. Far Red-labeled Raji (20,000 cells per well) were incubated in a 96-well round-bottom plate (Corning-3799) with isolated CD8 + T cells (5: 1 ratio of effector / target cells) and antibodies diluted in series at 37 ° C for 4 h. After incubation, 3 µM of propidium iodide (PI, Invitrogen-P3566) was mixed very well to identify dead cells. After 15 minutes, the cells were analyzed by flow cytometry using a FACSCanto II cytometer. Ab-mediated cytotoxicity can be defined as the percentage of PI positive target cells in Far Red positive target cells. EC50 of cytotoxicity was determined by Prism.

T cell activation assay Secreted TNFα and IFNγ cytokines

[0549] T cell activation was reflected by the amount of TNFα and IFNγ secreted into the supernatant. The procedure for isolating CD4 and CD8 positive T cells has been described in Section “Activation of T cells (intracellular labeling of TNFα and IFNγ cytokines)”. The mixture of human Raji B cells (2 x 10 4 cells / well), CD4 or CD8 T cells (1 x 105 cells / well) and antibodies was matched at 37 ° C for 24 h. The supernatant was collected followed by centrifugation of the reaction mixture at 1500 rpm for 5 min. The amount of TNFα and IFNγ in the supernatant was determined by human TNF ELISA (R & D-DY210) and human IFNγ ELISA (Capture Ab: Thermo Fisher - M700A, Detection Ab: Thermo Fisher - M701B, Standard Substance: PEROTECH - 300 -02), respectively.

[0550] The sandwich ELISA procedure was as follows. High-protein 96-well ELISA plates (ThermoFisher - 442404) were coated overnight at 4 ° C or at room temperature with 50 μL / well of capture antibody in carbonate-bicarbonate buffer (20 mM Na2CO3, 180 mM NaHCO3, pH 9.2) according to the kit specifications. All wells were washed three times with 300 μL of PBS / 0.5% Tween-20 (v / v) per well and all subsequent washing steps of the assay were performed equally. The wells were then blocked for one hour with PBS / 2% BSA (BovoGen Biologicals-BSAS) for TNFα and 100% casein (Pierce-37528) for IFNγ and then washed three times, followed by the binding of the collected supernatant or substance standard (50 μL / well) for 1 hour at room temperature and three washes afterwards. For the binding of the detection antibody, the corresponding antibodies diluted in PBS / 2% BSA for TNFα and 50% casein for IFNγ were added to the relevant wells and incubated at room temperature for two hours. The plates were washed three times before adding 50 μL of secondary antibody SA-HRP. The plates were incubated at room temperature for one hour, followed by six washes as described above. For the detection of binding, 50 μL of tetramethylbenzidine (TMB) substrate solution (Sigma-860336) was added to all wells for 10 minutes before stopping the reaction with 50 mL of 2M HCl. The amount of TNFα and IFNγ was determined by measuring the absorbance at OD450 using the SpectraMax® M5e microplate reader.

T cell activation assay - expression of surface marker CD25 and CD69

[0551] T cell activation was reflected by the CD25 and CD69 surface receptor labeling signals. The procedure for isolating CD4 and CD8 positive T cells has been described in Section “Activation of T cells (intracellular labeling of TNFα and IFNγ cytokines)”. The mixture of human Raji B cells (2 x 10 4 cells / well), CD4 or CD8 T cells (1 x 105 cells / well) and antibodies was matched at 37 ° C for 24 h. After washing once with 1% BSA, cell pellets were resuspended with labeling buffer containing FITC-labeled mouse human CD4 (BD-550628) or PerCPCy5.5-labeled mouse human CD8 (BD-560662 ), PE-labeled mouse anti-human CD69 (BD-560968) and APC-labeled mouse anti-human CD25 (BD-555434), followed by a 30 minute incubation at 4 ° C.

After two washes of cells, the percentage of cells positive for PE and APC in cells positive for FITC or PerCPCy5.5 was determined by flow cytometry.

Thermal stability (DSF)

[0552] The fusion temperature (Tm) of the antibodies was investigated using QuantStudio 7 Flex real-time PCR system (Applied Biosystems). 19 µL of the antibody solution was mixed with 1 µL of the SYPRO Orange (Invitrogen) 62.5x solution and transferred to a 96-well plate (Biosystems). The plate was heated from 26 ° C to 95 ° C at a rate of 0.9 ° C / min, and the resulting fluorescence data was collected. The negative derivatives of the fluorescence changes in relation to the different temperatures were calculated and the maximum value was defined as the melting temperature Tm. If a protein contained multiple split transitions, the first two Tm were identified and named Tm1 and Tm2. Data collection and Tm calculation were performed automatically by the operating software.

Serum stability

[0553] Fresh human blood was collected from selected donors and transferred to polystyrene tubes without anticoagulant. After 30 min of rest at room temperature, human blood was centrifuged at 4000 rpm for 10 minutes to collect the serum layer. The centrifugation step was repeated until the serum clarified. The antibodies were mixed under detection with the serum collected in the proportion of 1: 9, and the aliquots were collected at 37 ° C at the indicated times: 0 day, 1 day, 4 days, 7 days and 14 days. The samples were quickly frozen at different time points in liquid nitrogen and stored at -80 ° C until use. The samples were analyzed by FACS to assess the binding capacity on CD3 Jurkat T cells and CD19 Ramos B cells compared to the corresponding antibodies without serum treatment.

Affinity of binding to the Fcγ receptor by SPR

[0554] The antibody binding affinity to FcγRs was detected using Biacore T200 (or Biacore 8K). Each receiver was captured on a CM5 (GE) sensor chip immobilized with anti-his antibody. The antibodies in different concentrations were injected on the sensor chip at a flow rate of 30 µL / min for a 60 s association phase, followed by 60 s dissociation. The chip was then regenerated by 10 mM glycine (pH 1.5) after each binding cycle.

[0555] The sensograms of the buffer channel and reference surface (blank) were subtracted from the test sensograms. The experimental data were adjusted by the 1: 1 model using Langmiur analysis (for FcγRI) or steady state model (for other receptors). The molecular weight of 150 KDa was used to calculate the molar concentration of the antibodies.

Binding to C1q by ELISA

[0556] ELISA plates (Nunc) were coated with antibody samples at 3μg / mL overnight at 4 ° C. After blocking and washing, the C1q was gradually diluted from 600 μg / mL and incubated at room temperature for 2 hours. The plates were then washed and subsequently incubated with sheep anti-human C1q Ab-HRP for 1 hour. After washing, the TMB substrate was added and the interaction was interrupted by 2 M HCl. The absorbance at 450 nm was read using a microplate reader (Molecular Device).

FcRn binding affinity for SPR

[0557] The antibody binding affinity to FcRn was detected using Biacore T200 (or Biacore 8K). Each antibody was immobilized on the CM5 (GE) sensor chip. FcRn in different concentrations in running buffer (50 mM Na2HPO4 / NaH2PO4, 150 mM NaCl, 0.05% Tween 20, pH 6.0) were injected onto the sensor chip at a flow rate of 30 µL / min for a 60 s association phase, followed by 60 s of dissociation. The chip was then regenerated by PBS 1X (pH 7.4) after each binding cycle.

[0558] The sensograms of the buffer channel and reference surface were subtracted from the test sensograms. The experimental data were adjusted using the steady state model. A molecular weight of 45 KDa was used to calculate the molar concentration of FcRn.

Efficacy study in the murine Raji / PBMC model

[0559] Raji tumor cells (ATCC® CCL-86 ™) were maintained in vitro as a monolayer culture in RPMI-1640 supplemented with 10% fetal bovine serum, 100 U / ml penicillin and 100 µg / ml streptomycin at 37 ° C in an atmosphere of 5% CO2 in the air. Tumor cells were routinely subcultured twice a week. Cells that grow in an exponential growth phase were collected and counted for tumor inoculation.

[0560] Human PBMCs were isolated from heparinized whole blood using Ficoll-Paque Plus according to the manufacturer's instructions.

[0561] Each mouse was co-inoculated subcutaneously on the right flank with Raji tumor cells mixed with Matrigel and fresh PBMC in 0.2 mL of PBS in D0. The antibody injection was performed from D3 (i.v. twice a week x 4 times).

Preparation of test article Conc. Compounds Dosage Preparation mg / ml Isotype Control 0.031 ml of solution + 9.38 mg / ml 1.5 1.908 ml of PBS W3438-T3U4.E17-1.uIgG4.SP B: 0.138 ml of solution + 1.5 2.244 ml of PBS W3438-T3U4.E17-1.uIgG4.SP B1: 0.450 mL of B + 1.800 2.6 mg / mL 0.3 mL of PBS W3438-T3U4.E17-1.uIgG4.SP B2: 0.450 mL of B1 + 0.06 1.800 mL of PBS Outcomes and Measurements of tumors

[0562] The main outcome was to assess whether tumor growth could be slowed or whether mice could be cured. The tumor size was measured twice a week in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V = 0.5 ax b2, where a and b are the long and short diameters of the tumor, respectively . The T / C value (in percentage) is an indication of antitumor effectiveness.

[0563] The TGI was calculated for each group using the formula: TGI (%) = [1- (Ti-T0) / (Vi-V0)] × 100; Ti is the average tumor volume of a treatment group on a given day, T0 is the average tumor volume of the treatment group on the day the treatment starts, Vi is the average tumor volume of the vehicle control group on the same day with Ti, and V0 is the average tumor volume of the vehicle group on the day of the start of treatment.

Pharmacokinetics (PK), toxicity and immunogenicity in cynomolgus monkeys

[0564] 1 mg / kg of WBP3438 once per bolus intravenous administration was administered to a male cinomolgo monkey and a female cinomolgo monkey. The formulations were formulated in 20 mM NaAc-HAc, 7.0% (w / w) Sucrose, 0.02%

(w / v) of PS80, pH 5.0. PK blood samples were collected at pre-dose (Day-1), 0.25 h, 0.5 h, 1 h, 4 h, 8 h, 24 h, Day 3, Day 7, Day 14, Day 21 and Day 28. Anti-drug antibody (ADA) samples were collected in 3d, 14 d (312 h) and 28 d (480 h).

[0565] Serum concentrations of WBP3438 and ADA in serum samples were determined by ELISA. The serum concentration of WBP3438 in monkeys was subjected to a non-compartmental pharmacokinetic analysis using the Phoenix WinNonlin software (version 6.3, Pharsight, Mountain View, CA). The linear / log trapezoidal rule was applied to obtain the PK parameters.

[0566] Laboratory observations (cage-side) regarding health and general appearance, especially skin irritation were observed. The analysis of the whole blood sample for hematology (CBC) and the serum analysis for chemical detection were determined by the hematological (ADVIA2120) and chemical (HITACHI 7180) analyzer, respectively.

Results Generation of cynomolgo cell line expressing CD19

[0567] The expression of the CD19-expressing cinomolg cell line, WBP701.CHO-K1.cpro1.FL.C9, was detected using anti-CD19 antibody by flow cytometry. WBP701.CHO-K1.cpro1.FL.C9 showed high expression of monkey CD19 (Figure 37).

Generation and optimization of WuXiBody

[0568] Figure 1 shows schematic representations of the studied antibodies and formats. The T3 anti-CD3 antibody and the U4 anti-CD19 antibody were developed. The T3 constant region (CL and CH1) has been replaced by the TCR constant domain to develop a single light-heavy chain interface that is orthogonal to the regular antibody. T3 modified with TCR and native U4 together with the “knobs-into-holes” mutations in the Fc domain were used to develop the bispecific antibody format E17 and F16.

[0569] Light chain and variable heavy chain sequences of the anti-CD3 and anti-CD19 binding portions of W3438-T3U4.E17-1.uIgG4.SP and W3438- T3U4.F16-1.uIgG4.SP are provided below :

CAGGTGCAGCTTGTGCAGTCTGGGGCAGAAGTGAA GAAGCCTGGGTCTAGTGTCAAGGTGTCATGCAAGG CTAGCGGGTTCGCCTTTACTGACTACTACATCCAC TGGGTGCGGCAGGCTCCCGGACAAGGGTTGGAGTG

GATGGGATGGATCTCCCCAGGCAATGTCAACACAA DNA Sequence AGTACAACGAGAACTTCAAAGGCCGCGTCACCATT (SEQ ID NO: 353) ACCGCCGACAAGAGCACCTCCACAGCCTACATGGA VH of the GCTGTCCAGCCCTGGGGGGG

TTTGATTACTGGGGCCAGGGCACACTGGTGACAGT GAGCTCC

QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIH WVRQAPGQGLEWMGWISPGNVNTKYNENFKGRVTI String (SEQ TADKSTSTAYMELSSLRSEDTAVYYCARDGYSLYY ID NO: 352) FDYVG

GATATCGTGATGACCCAGAGCCCAGACTCCCTTGC TGTCTCCCTCGGCGAAAGAGCAACCATCAACTGCA AGAGCTCCCAAAGCCTGCTGAACTCCAGGACCAGG

AAGAATTACCTGGCCTGGTATCAGCAGAAGCCCGG CCAGCCTCCTAAGCTGCTCATCTACTGGGCCTCCA DNA sequence (SEQ ID NO: 355) CCCGGCAGTCTGGGGTGCCCGATCGGTTTAGTGGA TCTGGGAGCGGGACAGACTTCACATTGACAATTAG antibody VL anti-CD3 CTCACTGCAGGCCGAGGACGTGGCCGTCTACTACT W3438- GTACTCAGAGCCACACTCTCCGCACATTCGGCGGA T3U4.E17- GGGACTAAAGTGGAGATTAAG

1.uIgG4.SP DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTR & W3438- KNYLAWYQQKPGQPPKLLIYWASTRQSGVPDRFSG T3U4.F16- amino acids (SEQ SGSGTDFTLTISSLQAEDVAY

1.uIgG4.SP ID NO: 354) GTKVEIK

CAAATGCAGCTCGTCCAGTCTGGACCTGAAGTGAA GAAGCCCGGGACATCCGTCAAGGTCTCATGTAAGG CTAGCGGGTACGCATTCACTTCCTACAACATGTAC

TGGGTGCGCCAGGCCAGAGGACAGAGGTTGGAGTG DNA Sequence GATCGGCTACATCGACCCATACAACGCCGATACTA (SEQ ID NO: 368) CCTACAATCAGAAGTTTAAAGGGCGGGGGGGGGGGGGGGGC

ACTACTGCCTGACAACAGCCTATGCCATGGACTAT TGGGGCCAGGGCACACTTGTGACTGTGAGCAGT

QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMY WVRQARGQRLEWIGYIDPYNADTTYNQKFKGRVTI String (SEQ TRDMSTSTAYMELSSLRSEDTAVYYCLTTAYAMDY ID NO: 367) WGG

GACATCCAGCTCACCCAATCCCCTTCTTTCCTCTC CGCAAGTGTCGGAGATAGGGTGACTATCACCTGCT

CAGCTTCTTCAACCGTGAACTACATGCATTGGTAC VL of the CAGCAGAAGCCCGGGAAAGCCCCAAAGCTGCTGAT antibody sequence CTACAGCACCTCCAATCTGGCCAGTGGAGTGCCAA (SEQ ID NO: 370) anti-CD19 GCCGGGGTG

ACTTTGACAATTAGCAGCCTTCAGCCTGAGGACTT TGCCACATATTACTGTCACCAGTGGTCCAGCTACC CCTACACATTCGGGCAGGGCACAAAGCTGGAGATT THE AG

DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWY String

QQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEF amino acids (SEQ TLTISSLQPEDFATYYCHQWSSYPYTFGQGTKLEI ID NO: 369) K

[0570] The complete sequences of W3438-T3U4.E17-1.uIgG4.SP and W3438- T3U4.F16-1.uIgG4.SP are provided below: Antibody Chain Sequences

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQ

QKPGQPPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQA T3-LC (SEQ ID EDVAVYYCTQSHTLRTFGGGTKVEIKPDIQNPDPAVYQLRDSK NO: 12) SSDKSVCLFTDFDSQTDVY

NSAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQ GLEWMGWISPGNVNTKYNENFKGRVTITADKSTSTAYMELSSL RSEDTAVYYCARDGYSLYYFDYWGQGTLVTVLEDLKNVFPPEV AVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHS

GVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQ T3-HC (SEQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPE ID NO: 25) FLGGPSVFLFPPKPKDTLMISRTPEVTCVV

WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQ

VSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF W3438- LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK T3U4.E17- DIQLTQSPSFLSASVGDRVTITCKASVN

1.uIgG4.SP KLLIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYY U4-LC (SEQ ID NO: CHQWSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA 23) SVVCLLNNFYQREKESQ

SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQ RLEWIGYIDPYNGDTTYNQKFKGRVTITRDMSTSTAYMELSSL RSEDTAVYYCLTTAYAMDYWGQGTLVTVSSASTKGPSVFPLAP

CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV U4-HC LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE (SEQ ID NO: SKYGPPCPPCPAPEFLGPSVFLFPPVTFVTFVTFPVTFPVTFKVTFKVTQVTFKVTTT

LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV CTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHN

HYTQKSLSLSLGK Antibody Chain Strings

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQ

QKPGQPPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQA T3-LC (SEQ EDVAVYYCTQSHTLRTFGGGTKVEIKPDIQNPDPAVYQLRDSK ID NO: 12) SSDKSVCLFTDFDSQTDVY

NSAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS W3438-

QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQ T3U4.F16- GLEWMGWISPGNVNTKYNENFKGRVTITADKSTSTAYMELSSL

1.uIgG4.SP RSEDTAVYYCARDGYSLYYFDYWGQGTLVTVLEDLKNVFPPEV T3-HC (SEQ AVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHS ID NO: 25) GVCTDPQPLKEQPALQDSATFRL

VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN

Antibody Chain Sequences

WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQ VSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK DIQLTQSPSFLSASVGDRVTITCSASSTVNYMHWYQQKPGKAP

KLLIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYY U4-LC (SEQ CHQWSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA ID NO: 23) SVVCLLNNFYPREAKVQWKVDNESTE

SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC QMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNMYWVRQARGQ RLEWIGYIDPYNGDTTYNQKFKGRVTITRDMSTSTAYMELSSL RSEDTAVYYCLTTAYAMDYWGQGTLVTVSSASTKGPSVFPLAP CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVG GGGSGGGGSQMQLVQSGPEVKKPGTSVKVSCKASGYAFTSYNM

YWVRQARGQRLEWIGYIDPYNGDTTYNQKFKGRVTITRDMSTS U4-HC (SEQ TAYMELSSLRSEDTAVYYCLTTAYAMDYWGQGTLVTVSSASTK ID NO: 27) GPSVFPLAPCSRSTSESTAALGCLVKDYF

SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK

[0571] Production of W3438-T3U4.F16-1.uIgG4.SP

[0572] W3438-T3U4.F16-1.uIgG4.SP antibody expression titer is greater than 90 mg / L through transient expression. After two-step purification, the purity of W3438-T3U4.F16-1.uIgG4.SP reaches 97.5% (SEC-HPLC, Figure 39).

W3438-T3U4.F16-1.uIgG4.SP migrates with the apparent molecular mass of 75 kDa, 55 kDa and 25 kDa in SDS-PAGE under reducing conditions, which corresponds to the two heavy chains and two light chains. The two light chains can overlap due to similar molecular weights. The antibody migrates with the apparent molecular mass of 200 kDa under non-reducing condition, indicating the initial bispecific molecule (Figure 38).

Production of W3438-T3U4.E17-1.uIgG4.SP

[0573] W3438-T3U4.E17-1.uIgG4.SP antibody expression titer is greater than 100 mg / L through transient expression. After two-step purification, the purity of W3438-T3U4.E17-1.uIgG4.SP reaches 95% (SEC-HPLC, Figure 41). W3438-T3U4.E17-1.uIgG4.SP migrates with the apparent molecular mass of 54 kDa,

56 kDa and 25 kDa in SDS-PAGE under reducing conditions, which corresponds to two heavy chains and two light chains. The two light chains can overlap due to similar molecular weights. The antibody migrates with the apparent molecular mass of 150 kDa under non-reducing condition, indicating the intact bispecific molecule (Figure 40).

Link to target

[0574] The binding of W3438-T3U4.E17-1.uIgG4.SP to CD19 and CD3 was tested in Ramos and Jurkat cells by flow cytometry (Figures 42A-42B). The antibody W3438-T3U4.E17-1.uIgG4.SP showed strong binding activity to Ramos and Jurkat cells, with EC50 values of 15.6 nM and 47 nM, respectively.

[0575] The binding of W3438-T3U4.F16-1.uIgG4.SP to CD19 and CD3 was tested in Ramos and Jurkat cells by flow cytometry (Figures 43A-43B). The W3438-T3U4.F16-1.uIgG4.SP antibody showed strong binding activity to Ramos and Jurkat cells, with EC50 values of 1.8 nM and 19.3 nM, respectively.

Cross-linking between species

[0576] The binding of W3438-T3U4.E17-1.uIgG4.SP to CD19 of cinomolgo was tested in cell WBP701.CHO-K1.cpro1.FL.C9 (cell that expresses CD19) by flow cytometry (Figure 44) . EC50 binding was 26 nM. The binding of W3438-T3U4.E17-1.uIgG4.SP to cinomolgo CD3 was tested using W331- cynoPro1.ECD.His (cinomolg CD3 epsilon protein) by ELISA (Figure 45). The EC50 binding was 0.04 nM.

Affinity to target cells

[0577] The binding affinity of W3438-T3U4.E17-1.uIgG4.SP to human CD19 and CD3 was tested in Ramos and Jurkat cells by flow cytometry. Free IgG / IgG bound against bound IgG was plotted in Figures 46A and 46B.

The adjusted KD values of binding to CD19 and CD3 were 23 nM and 9.0 nM, respectively.

Double binding to target cells

[0578] The activity of W3438-T3U4.E17-1.uIgG4.SP to bind CD3 T cell and CD19 B cell was tested using pre-labeled Jurkat Raji cells by flow cytometry (Figures 47A-47B). Q2 illustrates the bridged or linked Jurkat and Raji cell population. In comparison with the negative control, approximately 18% of the cells were ligated via the bispecific antibody W3438-T3U4.E17-1.uIgG4.SP.

Cytotoxicity assay

[0579] The cytotoxic activity of W3438-T3U4.E17-1.uIgG4.SP was evaluated using CD8 + T cells and Raji cells. W3438-T3U4.E17-1.uIgG4.SP induced rapid and effective cell lysis after 4 hours of incubation (Figure 48A) with an EC50 value of 15 nM. The maximum percentage of cell death was 90%.

[0580] The cytotoxic activity of W3438-T3U4.F16-1.uIgG4.SP was assessed using CD8 + T cells and Raji cells. W3438-T3U4.F16-1.uIgG4.SP induced rapid and effective cell lysis after 4 hours of incubation (Figure 48B) with an EC 50 value of 3.2 nM. The maximum percentage of cell death was 90%.

Target-specific T cell activation

[0581] W3438-T3U4.E17-1.uIgG4.SP was investigated in assays that indicated the activation of T cells through the activation markers CD69 and CD25 in the presence or absence of target cells CD19 +. The results demonstrate that W3438-T3U4.E17-1.uIgG4.SP induces the expression of CD25 and CD69 activation markers of T cells in a dose-dependent manner only in the presence of target CD19 + cells (Figures 49A-49D). When cell B is absent, expression of CD25 and CD69 was not observed in the subsets of CD4 + and CD8 + T cells.

[0582] W3438-T3U4.E17-1.uIgG4.SP has also been investigated in T cell activation assays for cytokine release in the presence or absence of CD19 + target cells. The results demonstrated that W3438-T3U4.E17-1.uIgG4.SP induces the release of IFNγ and TNFα in a dose-dependent manner only in the presence of target CD19 + cells (Figures 50A-50D). When cell B is absent, no IFNγ and TNFα were detected in the CD4 + and CD8 + T cell subsets.

Thermal stability

[0583] The thermal stability of W3438-T3U4.E17-1.uIgG4.SP was investigated using real-time PCR. Tm1 and Tm2 of W3438-T3U4.E17-

1.uIgG4.SP are 60.2 ° C and 72.7 ° C.

Serum stability

[0584] W3438-T3U4.E17-1.uIgG4.SP was incubated in serum at 37 ° C for 14 days.

The binding activity of the antibody incubated for 0, 1, 4, 7 and 14 days was detected by flow cytometry. The results showed that the binding activity of W3438-T3U4.E17-1.uIgG4.SP to both CD3 and CD19 cells was unchanged after incubation in human serum for 14 days (Figures 51A-51B).

Connection to the Fcγ receptor

[0585] W3438-T3U4.E17-1.uIgG4.SP binding activity to FcγRI, FcγRIIa (H167), FcγRIIa (R167), FcγRIIb, FcγRIIIa (F176), FcγRIIIa (V176) and FcγRIIIb were investigated by. The affinities are summarized in Table 39. W3438- T3U4.E17-1.uIgG4.SP showed a typical human IgG4 binding affinity for all Fcγ receptors.

TABLE 39. Affinity of W3438-T3U4.E17-1.uIgG4.SP to the Fc receptor by

SPR Receiver Fc KD (M) FcγRI 9,79E-09 FcγRIIa (H167) 2,05E-05 FcγRIIa (R167) 1,58E-05 FcγRIIb 2,41E-05 FcγRIIIa (F176) 2,93E-05 FcγRIIIa (V176) 1.40E-05 FcγRIIIb> 4.10E-05

[0586] The antibody binding activity to C1Q was tested by ELISA.

W3438-T3U4.E17-1.uIgG4.SP showed no sign of binding in the ELISA (Figure 52), and the control human IgG1 antibody showed a sign of normal binding.

Connection to FcRn

[0587] The binding of W3438-T3U4.E17-1.uIgG4.SP to FcRn was tested by SPR at pH 6.0. Affinity was adjusted to 2.58 µM, which is a typical human IgG4 affinity for FcRn.

Efficacy study in the PBMC / Raji xenograft model

[0588] In this study, the antitumor efficacy of W3438-T3U4.E17-1.uIgG4.SP in the humanized model of mixed PBMC containing Raji cell in NOG mice was investigated. The tumor growth curve is shown in Figure 53.

[0589] At D14, the average tumor size of the isotype-controlled treatment group reached 342 mm3. Treatment with 1.5 mg / kg and 0.5 mg / kg of W3438-T3U4.E17-1.uIG4.SP produced significant antitumor activity. The average tumor size was, respectively, 78 mm3 (T / C = 23.0%, TGI = 93.9%, p = 0.016) and 75 mm3 (T / C = 22.0%, TGI = 95.3 %, p = 0.014) and the tumor of an animal in the high-dose group was eradicated. W3438-T3U4.E17-1.uIgG4.SP at a very low dose (0.06 mg / kg) did not show antitumor activity.

Pharmacokinetics (PK) of WuXiBody in cinomolgo monkey

[0590] The concentration of W3438-T3U4.E17-1.uIgG4.SP in the cinomolgo serum was tested by ELISA (Figure 54). The calculated PK parameters were listed in Table 40. The half-life of W3438-T3U4.E17-1.uIgG4.SP for a single IV injection at 1 mg / kg was 152 hours. W3438-T3U4.E17-1.uIgG4.SP showed a much longer half-life in monkeys than blinatumomab, which has a very short half-life (1.5-2.6) in chimpanzees (evaluation report European Medicines Agency EMA / CHMP / 469312/2015).

TABLE 40. P34 in cinomolgo of W3438-T3U4.E17-1.uIgG4.SP

PK parameter W3438-T3U4.E17-1.uIgG4.SP C0 (μg / mL) 60.4 T1 / 2 (h) 152 Vdss (L / kg) 0.0513 Cl (mL / min / kg) 0.00462 AUC0-last (h * μg / mL) 3552 AUC0-inf (h * μg / mL) 3708 MRT0-last. (h) 157 MRT0-inf (h) 187 Toxicity

[0591] All monkeys tolerated the drug throughout the study.

No adverse effects were observed during the study's life phase. There was no evident change in food consumption and weight. The parameters for Hematology and Clinical Chemistry, including AST, ALT, WBC, HGB and HCT were, in general, within the reference range.

Immunogenicity

[0592] Results of immunogenicity tests W3438-T3U4.E17-

1.uIgG4.SP are shown in Figures 55A-55B. The anti-drug antibody (ADA) titers against W3438-T3U4.E17-1.uIgG4.SP in monkey serum 3, 14 and 28 days after the dose did not show any significant difference in relation to the pre-dose. Therefore, the single IV injection of W3438-T3U4.E17-1.uIgG4.SP at 1 mg / kg did not appear to be immunogenic in monkeys.

EXAMPLE 18: WuXiBody Anti-CTLA-4 x PD-1 Bispecific Basics

[0593] Cancer immunotherapy has become an area of research for the treatment of cancer. Protein 4 associated with cytotoxic T lymphocytes (CTLA-4) is one of the validated targets of the immunological check point. After activation of T cells, CTLA-4 is rapidly expressed in these T cells, usually within one hour of antigen involvement with the TCR. CTLA-4 can inhibit T cell signaling through competition with CD28, which mediates a well-characterized T cell co-stimulatory signal. The binding of CD28 to its ligands CD80 (B7-1) and CD86 (B7-2) in antigen presenting cells leads to the proliferation of T cells, inducing the production of interleukin-2 and anti-apoptotic factors. Because the binding affinity of CTLA-4 to CD80 and CD86 is much higher than that of CD28, CTLA-4 can compete with CD28 for binding to CD80 and CD86, leading to the suppression of T cell activation. induced in activated T cells, CTLA-4 is expressed constitutively on the surface of regulatory T cells (Treg), suggesting that CTLA-4 may be necessary for contact-mediated suppression and be associated with the production of immunosuppressive cytokines by Tregs, such as such as the transforming growth factor beta and interleukin-10.

[0594] CTLA-4 blockade can induce tumor regression, as demonstrated in several clinical and preclinical studies. Two antibodies against CTLA-4 are in clinical development. Ipilimumab (MDX-010, BMS-734016), a fully human anti-CTLA-4 monoclonal antibody of the IgG1-kappa isotype, is an immunomodulatory agent that has been approved as a monotherapy for the treatment of advanced melanoma. The proposed mechanism of action for ipilimumab is the interference in the interaction of CTLA-4, which is expressed in a subset of activated T cells, with the CD80 / CD86 molecules in cells presenting professional antigens. This results in the potentiation of T cells due to the blocking of inhibitory modulation of T cell activation promoted by the CTLA-4 and CD80 / CD86 interaction. The resulting activation of T cells, proliferation and infiltration of lymphocytes in tumors, leads to the death of tumor cells. The commercial dosage form is a concentration rate of 5 mg / mL for solution for infusion. Ipilimumab is also under clinical investigation for other types of tumors, including prostate and lung cancer. The second anti-CTLA-4 antibody in clinical development, Tremelimumab, was evaluated as monotherapy in melanoma and malignant mesothelioma.

[0595] Programmed Death-1 (PD-1, CD279) is a member of the CD28 family expressed in activated T cells and other immune cells. PD-1 involvement inhibits function in these immune cells. PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), both belonging to the B7 family. PD-L1 expression is inducible in a variety of cell types in lymphoid and peripheral tissues, while PD-L2 is more restricted to myeloid cells, including dendritic cells. The main role of the PD-1 pathway is to reduce the inflammatory immune response in tissues and organs.

[0596] Immunotherapy with a combination of monoclonal antibodies (mAb) that block CTLA-4 (Ipilimumab) and PD-1 (Nivolumab) has demonstrated clinical benefit beyond that observed with both mAb alone. The bispecific WuXiBody anti-CTLA-4 x PD-1 was developed to induce anti-tumor immunity by simultaneously blocking both checkpoint molecules.

Materials and methods General materials

[0597] The general research materials and their sources are listed in the table below. Materials Supplier Cat. Expi293F ™ Cells Thermo Fisher Cat. A14527 Transfection kit ExpiFectamine293 Thermo Fisher Cat. A14524 Expi293F ™ expression medium Thermo Fisher Cat. A1435101 Lipofectamine ™ Transfection Reagent Thermo Fisher Cat. 11668019 2000 FreeStyle ™ 293-F Thermo Fisher cells R79007 FreeStyle ™ 293 Thermo Fisher Cat. 12338002 CHO-S cells Thermo Fisher Cat. A1155701 FreeStyle ™ CHO GIBCO Cat. 12651014 Fetal Serum (SFB) Corning Cat. 35-076-CV Opti-MEM Thermo Fisher Cat. 31985070 Ni column GE healthcare Cat. 17-5247-01 Protein column A GE healthcare Cat. 17-5438-02 Superdex200 prep grade GE Healthcare Cat. 17-1043-01 HPLC-SEC TOSOH Cat. 0008541

Materials Supplier Cat. NuPAGE4% -12% Bis-Tris Gel Thermo Fisher Cat. NP0322BOX CTLA-4 Human: W316-hPro1.ECD.His Bell Biological Cat. 11159-H08H CTLA-4: W316-cpro1.ECD.his Bell Biological Cat. 90213-C08H Cinomolgo PD-1 Human: W305-hPro1.ECD.His Cinomolgo PD-1: W305-cynoPro1.ECD.His R&D Cat.R & D-8509-PD- 050 96-well casing plates for Nunc MaxiSorp, ELISA ThermoFisher 96-well U-bottom plates for Corning-COSTAR 3799

FACS Human PD-1 + cell line: W305- Own CHO-S.hPro1.C6 Cinomolgo PD-1 + cells: W305- Own 293F.cPro1.FL.Pool Human CTLA-4 + cell line: Own W316-293F. hPro1.FL Cell line CTLA-4 + from Cinomolgo: Own W316-293F.cPro1.FL.Pool Cell line CD80 + Human: W316- Own CHO-K1.hPro1L1.B9B11 Cell line CD86 + Human: W316- Own CHO-K1.hPro1L2 .A4A7 Human PD-1: W305-hPro1.ECD.mFc Own PD-1 Human: W305-hPro1.ECD.hFc Own Human PD-1: W305-hPro1.ECD.His Cinomolgo Own PD-1: W305-cPro1 .ECD.His Own Protein CynoPD-1.hFc Biological Bell 90311C02H Human CTLA-4: W316-hPro1.ECD.mFc Own Human CTLA-4: W316-hPro1.ECD.hFc Own CTLA-4 Human: W316-hPro1.ECD Cinomolgo's own CTLA-4: W316- Human's cPro1.ECD.His Human PDL1: W315-hPro1.ECD.mFc Own CynoPD-L1.hFc-Biotin Own W316.hPro1.ECD.hFc marked with Own Human CD80 Biotin : W316-hPro1L1.ECD.His Own WBP316-BMK1 (Ipilimumab) Own WBP305 BMK1 (nivolumab) rio WBP324-BMK1.IgG1.KDL Own Isotype Control: WBP332- Own

1.80.12.xAb.hIgG4 anti-IgG Fc Human goat marked Bethyl Laboratories A80-304P with HRP

Materials Supplier Thermo MA5-16859 HRP-labeled anti-human IgG Fc mouse HRH (CH2) goat anti-IgG Fc Bethyl Laboratories A90-231P labeled HRP Streptavidin labeled with HRP Life technologies SNN1004 anti-His mAb labeled biotin GenScript A00613 goat anti-human IgG labeled with Jackson 109-095-008

Jackson-tagged goat anti-human IgG FITC 109-115-098

Abcam 98716 labeled goat anti-IgG PE with FITC PE labeled streptavidin BD 554061 Ficoll-Paque Human Stemcell 07861 Miltenyi Biotec monocyte enrichment kit Biotec-130-050-201 CD4 + cell enrichment kit Stemcell 19052 Enrichment kit Miltenyi T cells 130-093-631 CD4 + CD25 + recombinant human R&D 215-GM recombinant human IL-2 SL PHARM IL-4; recombinant anti-IL-2 Ab R&D AG1815401; MAB602 Peprotech 300-02 Human Recombinant IFN-γ Standard Life technology M700A; M7001B H3-thymidine and MicroScint Perkin Elmer NET027001MC Bovine Fetal Serum (SFB) GIBCO 10100147 RPMI 1640 GIBCO 22400089 CVR Cytotoxicity Reagents DELFIA® Perkin Elmer AD0116 EuTDA Cell viability assay kit Promega G7573 luminescent CellTiter-Glo Calcein-AM Corning-354216 354216 Far red Invitrogen C34572 Soluble Antigen Generation

[0598] The DNA sequences encoding the human PD-1 extracellular domain sequence (Uniport No .: Q15116) were synthesized by Sangon Biotech (Shanghai, China) and subcloned into 6xhis modified pcDNA3.3 expression vectors in C-terminal end. Proteins from human CTLA-4, cinomolgo and mouse and PD-1 from mouse and cinomolgo were purchased from Sino Biological.

[0599] Expi293 cells (Invitrogen-A14527) were transfected with the purified expression vector pcDNA3.3. The cells were cultured for 5 days and the supernatant was collected for protein purification using the Ni-NTA column (GE Healthcare, 175248). The human PD-1 obtained was quantified (QC'ed) by SDS-PAGE and SEC, and then stored at -80 ° C.

Generation of reference antibodies

[0600] The DNA sequence encoding the variable region of the anti-CTLA-4 antibody (WBP316-BMK1), anti-PD-1 antibody (WBP305-BMK1) was synthesized by Sangon Biothech (Shanghai, China) and subcloned into vectors modified pcDNA3.4 expression cells with human IgG1 or human IgG4 (S228P) constant region. The anti-PD-1 antibodies WBP3055-1.153.7.uIgG4k and WBP3055-

1,103.11.uIgG4k were generated after immunization of rats with human PD-1 and mouse PD-1, and were converted to the IgG4 format (S228P). The DNA sequence encoding a bispecific anti-CTLA-4 x PD-1 reference antibody (WBP324-BMK1.IgG1.KDL) was synthesized.

[0601] Plasmids containing the VH and VL genes were cotransfected into Expi293 cells. The cells were cultured for 5 days and the supernatant was collected for protein purification using the protein A column (GE Healthcare, 175438) or the protein G column (GE Healthcare, 170618). The obtained antibodies were tested by SDS-PAGE and SEC and stored at -80 ° C.

Generation of cell lines expressing targets

[0602] Using lipofectamine 2000, CHO-S or 293F cells were transfected with the expression vector containing the genes encoding human PD-1 or complete mouse PD-1. The cells were grown in medium containing suitable selection markers. The high-expression human PD-1 stable cell line (WBP305.CHO-S.hPro1.C6) and the mouse high-expression stable PD-1 cell line (WBP305.293F.mPro1.B4) were obtained by dilution limiting.

Generation of bispecific anti-CTLA-4 / PD-1 antibodies

[0603] The W3248-U6T1.G25R-1.uIgG4.SP construct: DNA sequence encoding the anti-PD-1 heavy chain variable region, constant region 1, anti-CTLA- heavy chain variable region 4, beta constant region of the TCR and IgG4 constant regions 2 and 3 (S228P), linked from the 5 'to the 3' end, were cloned into a modified pcDNA3.3 expression vector. The DNA sequence encoding the variable region of the anti-CTLA-4 antibody light chain at the 5 'end of the TCR alpha constant region was cloned into another modified pcDNA3.3 expression vector. The anti-PD-1 light chain was cloned into the modified third pcDNA3.3 expression vector.

[0604] The W3248-U6T5.G25-1.uIgG4.SP construct: DNA sequence encoding anti-PD-1 heavy chain variable region, TCR beta constant chain region, anti heavy chain variable region -CTLA-4 and IgG4 constant region (S228P), linked from the 5 'to the 3' end, were cloned into a modified pcDNA3.3 expression vector. The DNA sequence encoding the variable region of the anti-PD-1 antibody light chain at the 5 'end of the TCR alpha constant region was cloned into another modified pcDNA3.3 expression vector.

The anti-CTLA-4 light chain was cloned into the modified third pcDNA3.3 expression vector.

[0605] Relevant strings from W3248-U6T1.G25R-1.uIgG4.SP are provided below:

SYELTQPLSVSVALGQTARITCGGDNIGNKKVVHWYQQKPGQAP String

VLVIYRDSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYY Amino Acids LC1 CQVWDSIWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKA (SEQ ID NO: TLVCLISDFYPGAVTVAWKADSSPTKKTSSKTQKTTKKTSSKTTKKTTKKTTKT

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQ String

APRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAV Amino Acids LC2 YYCQQYGSSPWTFGQGTKVEIKPDIQNPDPAVYQLRDSKSSDK (SEQ ID NO: SVCLFTDFDSQTQVSQSKDSDVFQSQSQSQSQSQSKSDQY

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK GLEWVSTITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAKNRAGEGYFDYWGQGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSS

YTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDN String

SKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVL amino acids

HC EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVEL (SEQ ID NO: SWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATF 414) WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQSA

GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLG

[0606] Relevant strings from W3248-U6T5.G25-1.uIgG4.SP are provided below:

SYELTQPLSVSVALGQTARITCGGDNIGNKDVHWYQQKPGQAP VLVIYRDSNRPSGIPEGFSGSNSGNTATLTISRAQAGDEADYY amino acid sequence LC1 CQVWDSIWVFGGGTKLTVLPDIQNPDPAVYQLRDSKSSDKSVC (SEQ ID NO: 415) LFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS

QKSDFACANAFQNSIIPEDTFFPSPESS

EIVLTQSPDFQSVTPKEKVTITCSANSALSYMYWYQQKPDQSP KLWVHGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYY LC2 Amino Acids CHHWSNTQWTFGGQTQVQKQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQT

SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGK GLEWVSTITGGGGSIYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAKNRAGEGYFDYWGQGTLVTVLEDLKNVFPPEV AVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHS GVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQ VQFYGLSENDEWTQDRAKPVTQIVSAEAWGRGGGGSGGGGSQV

QLVQSGAEVKKPGSSVKVSCKASGYTFTNYFMNWVRQAPGQGL EWMGRVDPEQGRADYAEKFKKRVTITADKSTSTAYMELSSLRS HC String

VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLG

[0607] For both bispecific antibodies, a heavy chain expression vector and two light chain expression vectors were cotransfected into Expi293 cells (ThermoFisher-A14527) according to the manufacturer's instructions.

Five days after transfection, supernatants were collected and purified using protein A column (GE Healthcare-17543802) and exclusion chromatography for additional size (GE Healthcare-17104301). The concentration of antibodies was measured by Nano Drop. The low level of endotoxin was confirmed using the endotoxin detection kit (GenScript-L00350), and the endotoxin level of the two bispecific antibodies was less than 10 EU / mg. Protein purity was assessed by SDS-PAGE and HPLC-SEC.

In Vitro Characterization Differential scanning fluorimetry (DSF)

[0608] A DSF assay was performed using the 7500 Fast real-time PCR system (Applied Biosystems). Briefly, 19 µL of bispecific antibody solution was mixed with 1 µL of SYPRO Orange 62.5x solution (TheromFisher-S6650) and added to a 96-well plate. The plate was heated from 26 ° C to 95 ° C at a rate of 2 ° C / min and the resulting fluorescence data was collected. The data were automatically analyzed by your operating software and Th was calculated considering the maximum value of the negative derivative of the resulting fluorescence data in relation to the temperature. Ton can be roughly determined as the temperature of the negative derivative graph that starts to decrease from a pre-transition baseline.

Binding to human PD-1 by FACS

[0609] Modified cells expressing human PD-1, W305-CHO- S.hPro1.C6, were plated at 1 x 105 cells / well in 96-well U-bottom plates (Costar 3799). Antibodies with 3.16-fold titration in DPBS with 1% 200 nM BSA to 0.002 nM were added to the cells. The plates were incubated at 4 ° C for 1 hour. After washing, 100 µL of 1: 125 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ° C for 1 hour. The binding of antibodies to cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Connection to PD-1 cinomolgo by FACS

[0610] Modified cells expressing cinomolg PD-1, W305- 293F.cynoPro1.FL.pool, were plated at 1 x 105 cells / well in 96-well U-bottom plates (Costar 3799). Antibodies titrated 4 times in DPBS with 1% BSA from 40 µg / ml to 0.0001526 µg / ml were added to the cells. The plates were incubated at 4 ° C for 1 hour. After washing, 100 µL of goat anti-human antibody labeled with PE diluted 1: 150 (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ° C for 1 hour. The binding of antibodies to cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Binding to human CTLA-4 by FACS

[0611] Modified cells expressing human CTLA-4, W316- 293F.hPro1.FL, were plated at 1 x 105 cells / well in 96-well U-bottom plates (Costar 3799). Antibodies with 3.16-fold titration in DPBS with 1% 200 nM BSA to 0.002 nM were added to the cells. The plates were incubated at 4 ° C for 1 hour. After washing, 100 µL of 1: 125 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ° C for 1 hour. The binding of antibodies to cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Binding to CTLA-4 of cinomolgo by FACS

[0612] Modified cells expressing human CTLA-4, W316- 293F.cynoPro1.F1.pool, were plated at 1 x 105 cells / well in 96-well U-bottom plates (Costar 3799). Antibodies titrated 4 times in DPBS with 1% BSA of 40 µg / ml to 0.00004 µg / ml were added to the cells. The plates were incubated at 4 ° C for 1 hour. After washing, 100 µL of 1: 150 diluted PE-labeled goat anti-human antibody (Jackson 109-115-098) was added to each well and the plates were incubated at 4 ° C for 1 hour. The binding of antibodies to cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Double binding of hPD-1 and hCTLA-4 by ELISA

[0613] In order to test whether bispecific antibodies could bind to hPD-1 and hCTLA-4, an ELISA assay was developed as below. A 96-well ELISA plate (Nunc MaxiSorp, ThermoFisher) was coated overnight at 4 ° C with 0.5 µg / ml antigen-1 (hPD-1-ECD, W305-hPro1.ECD.mFc) in buffer carbonate-bicarbonate. After a 1 hour blocking step with 2% (w / v) bovine serum albumin (Pierce) dissolved in PBS, serial dilutions of the different bispecific antibodies PD-1 × CTLA-4 in PBS containing 2% BSA were incubated plates for 1 hour at room temperature. After incubation, the plates were washed three times with 300 μL per well of PBS containing 0.5% (v / v) Tween 20. 0.5 μg / mL antigen-2 (hCTLA-4-ECD, W316- hPro1.ECD.hFc.Biotin) was added to the plates and the mixture was incubated for 1 hour. After three washes of the plates, Streptavidin-HRP (Life Technologies, # SNN1004) (diluted 1: 20000) was added and incubated on the plates for 1 hour at room temperature. After six washes with 300 µL per well of PBS containing 0.5% (v / v) Tween 20, 100 µL of the substrate tetramethylbenzidine (TMB) was added for detection per well. The reaction was stopped after approximately 5 minutes, by adding 100 µL per well of 2 M HCl. The absorbance of the wells was measured at 450 nm with a multi-wall plate reader (SpectraMax® M5e).

Double binding to hPD-1 and hCTLA-4 by FACS

[0614] To test whether bispecific antibodies could bind to both hPD-1 and hCTLA-4, a FACS assay was developed as below. Modified cells expressing human PD-1 and CTLA-4, W305-CHO-S.hPro1.C6 and W316-293F.hPro1.F1, were stained with 50 nM calcein-AM (Corning-354216) and 20 nM Far Red (Invitrogen-C34572), respectively, for 20 minutes at 37 ° C. After two washes with 1% (w / v) bovine serum albumin (Pierce) dissolved in PBS, mixed hPD-1 (5E4) and hCTLA-4 (5E4) cells were plated at 1 x 10 5 cells / well in plates 96 U-bottom wells (Costar 3799). After removing the supernatant, antibodies diluted in series 3 x with DPBS and 1% BSA of 7.5 nM to 0.83 nM were added to the cells. The plates were incubated at 4 ° C for 1.5 hours. The cells were tested by flow cytometry and the percentage of double positive cells was analyzed by FlowJo.

Competitive FACS for human PD-1

[0615] To test whether bispecific antibodies could block the binding of hPD-L1 to hPD-1 protein, a competitive FACS was performed. Briefly, modified cells expressing human PD-1, W305-CHO-S.hPro1.C6 (self), were plated at 1 x 105 cells / well in 96-well U-bottom plates, 200 nM to 0.002 nM PD -L1 human coupled to 5 µg / mL of human PD-L1 protein W315-hPro1.ECD.mFc were added to the cells. The plates were incubated at 4 ° C for 1 hour. After washing, the binding of W315-hPro1.ECD.mFc to the human PD-1 expressed by the cell was detected by the FITC-labeled goat anti-mouse antibody (abcam 98716 1: 125). The competitive binding of antibodies to cells was tested by flow cytometry and the mean fluorescence intensity (MFI) was analyzed by FlowJo.

Blocking the binding of human CTLA-4 / cinomolgo to human CD80

[0616] ELISA was used to test whether bispecific antibodies could block hCTLA-4 binding to hCD80 protein. Briefly, 96-well flat bottom plates (Nunc MaxiSorp, ThermoFisher) were pre-coated with 0.5 μg / mL of W316-hPro1.ECD.hFc overnight at 4 ° C. After blocking with 2% BSA, 100 μL of antibodies with a 3.16-fold titration of 400 nM to 0.04 nM Abs, coupled to 0.5 μg / mL of human CD80 protein W316-hPro1L1.ECD, were pipetted into each well and incubated for 1 hour at room temperature. After incubation, the plates are washed 3 times with 300 μL per well of PBS containing 0.5% (v / v) of Tween 20. 100 µL of 0.5 μg / mL of biotin-labeled anti-His mAb (GenScript -A00613) were added to the plate per well and incubated for 1 hour. After 6 washes, the binding of W315-hPro1L1.ECD.His to WBP316-hPro1.ECD.hFc was detected by Streptavidin-HRP (Lifetechnologies, # SNN1004) (diluted 1: 20000). The color was developed by making available 100 μL of TMB substrate and then interrupted by 100 μL of 2N HCl. The absorbance was read at 450 nm using a microplate spectrophotometer (SpectraMax® M5e).

[0617] Competitive FACS was used to test whether antibodies could block the binding of human CTLA-4 or cinomolg to hCD80 on the cell surface. Briefly, CHO-K1 cells expressing human CD80 were added to each well of a 96-well plate (Costar 3799) at 1 x 10 5 per well and centrifuged at 1500 rpm for 4 minutes at 4 ° C before removing the supernatant . Serial dilutions of test antibodies, positive and negative controls were mixed with biotinylated human CTLA-4.ECD.hFc. Due to the different density of ligands on the cell surface, 0.066-0.037 µg / ml hCTLA-

4.ECD.hFc-Biotin was used for cells that express human CD80. Then, mixtures of antibody and CTLA-4 were added to the cells and incubated for 1 hour at 4 ° C. The cells were washed twice with 200 ml of FACS wash buffer (DPBS containing 1% BSA). Streptavidin PE (BD Pharmingen- 554061) diluted 1 to 600 in FACS buffer was added to the cells and incubated at 4 ° C for 1 hour. Additional washing steps were performed twice with 200 μL of FACS wash buffer, followed by centrifugation at 1500 rpm for 4 minutes at 4 ° C. Finally, cells were resuspended in 100 μL of FACS wash buffer and fluorescence values were measured by flow cytometry and analyzed by FlowJo.

Affinity to CTLA-4 and PD-1

[0618] SPR technology was used to measure the on-rate constant (ka) and off-rate constant (kd) of antibodies to CTLA-4 or PD-1 ECD. The affinity constant (KD) was consequently determined.

[0619] Biacore T200, CM5 S-series sensor chip, amine coupling kit and 10x HBS-EP were purchased from GE Healthcare. Goat anti-human IgG Fc antibody was purchased from the Jackson ImmunoResearch Lab (catalog number 109-005-098). In the immobilization step, the activation buffer was prepared by mixing 400 mM EDC and 100 mM NHS immediately before injection. The CM5 sensor chip was activated for 420 s with the activation buffer. 30 µg / mL of goat anti-human IgG Fcγ antibody in 10 mM NaAc (pH 4.5) was then injected into the Fc1-Fc4 channels for 200 s at a flow rate of 5 µL / min. The chip was deactivated with 1 M ethanolamine-HCl (GE). Then, the antibodies were captured on the chip. Briefly, 4 μg / mL of antibodies in running buffer (HBS-EP +) were injected individually into the Fc3 channel for 30 s at a flow rate of 10 μL / min.

Eight different concentrations (20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 nM) of the CTLA-4 or PD-1 ECD analyte and reference running buffer (blank) were injected in order in the Fc1-Fc4 channels at a flow rate of 30 μL / min for an association phase of 120 s, followed by the dissociation phase of 2400 s. The regeneration buffer (10 mM glycine, pH 1.5) was injected at 10 µL / min for 30 s after each dissociation phase.

Stability in human serum

[0620] The antibodies were incubated in fresh human serum at 37 ° C. At the indicated time points, an aliquot of sample treated with serum was removed from the incubator and quickly frozen in liquid nitrogen, and then stored at -80 ° C until ready for a double bond assay by ELISA. The frozen samples were quickly thawed immediately before the stability test. Briefly, the plates were pre-coated with 0.5 μg / mL of hCTLA4.ECD.hFc (internally) at 4 ° C overnight. After 1 hour of blocking, the test antibodies were added to the plates in various concentrations. The plates were incubated at room temperature for 1 hour. After incubation, the plates were washed three times with 300 μL per well of PBS containing 0.5% (v / v) Tween

20. Then, 0.1 μg / mL of hPD-1-ECD. biotin was added to the plates and the mixture was incubated for 1 hour. After three washes of the plates, Streptavidin-HRP (Life Technologies, # SNN1004) (diluted 1: 20000) was added and incubated on the plates for 1 hour at room temperature. After six washes with 300 µL per well of PBS containing 0.5% (v / v) Tween 20, 100 µL of the substrate tetramethylbenzidine (TMB) was added for detection per well. The reaction was stopped after approximately 5 minutes, by adding 100 µL per well of 2 M HCl. The absorbance of the wells was measured at 450 nm with a multi-wall plate reader (SpectraMax® M5e).

Results Expression and purification of bispecific antibodies

[0621] The purity of bispecific antibodies was greater than 90%, analyzed by both SDS-PAGE (Figure 56 A) and SEC-HPLC (Figure 56B).

WuXiBody DSF

[0622] The DSF was used to measure WuXiBody Tm. As shown in Figure 57, W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP have Th1 at 60.8 and 63.4 ° C, respectively.

Binding to human and cinomolgo PD-1

[0623] Bispecific antibodies can bind to human PD-1 (Figure 58) and cinomolgo PD-1 (Figure 59). The human PD-1 binding activity of W3248-U6T1.G25R-1.uIgG4.SP was slightly better than WBP3248-U6T5.G25-1- uIgG4.SP by FACS. W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1- uIgG4.SP have affinity for human PD-1 at 1.24 nM and 1.32 nM, respectively (Figure 62).

Binding to human and cinomolgo CTLA-4

[0624] Purified bispecific antibodies bound to human CTLA-4, as tested by FACS (Figure 60). The two bispecific antibodies also bound to cinomolgo CTLA-4 (Figure 61). W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP have an affinity for human CTLA-4 at 0.0356 nM and 0.357 nM, respectively (Figure 62).

Simultaneous connection to CTLA-4 and PD-1

[0625] To test whether bispecific antibodies can bind to both targets, ELISA and FACS were used. In the ELISA, human PD-1 was coated on the plate.

After addition of bispecific antibodies, biotinylated CTLA-4 was used to detect bound bispecific antibodies. As shown in Figure 66, W3248- U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP can bind to PD-1 and CTLA-4 with EC50 from 0.1072 to 0, 0710 nM, comparable to a bispecific reference antibody WBP324BMK1 (EC50 = 0.0599 nM). In FACS, both W3248- U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP can simultaneously bind to PD-1 + and CTLA-4 + cells (Figure 67).

Blocking the binding of human CTLA-4 or cinomolg to the binding of CD80

[0626] Competitive FACS was used to test the CTLA-4 blockade of bispecific antibodies with its CD80 ligand. W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP blocked the binding of CTLA-4 to CD80 with IC 50 of 4,300 and 0,7581 nM (Figure 64). Likewise, bispecific antibodies can also block the binding of cinomolgo CTLA-4 to human CD80 + cells (Figure 65).

Blocking the connection of PD-1 to its ligand

[0627] A competitive FACS was used to test the blocking of bispecific antibodies from PD-1 with its ligand, PD-L1. W3248-U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP blocked the binding of PD-1 to PD-L1 with an IC50 of 1.670 nM and 1.917 nM (Figure 63).

Serum stability

[0628] The two bispecific antibodies were incubated in human serum at 37 ° C for 14 days, and their double binding to human CTLA-4 and PD-1 was measured by ELISA. As shown in Figures 68A and 68B, the double bonding of W3248- U6T1.G25R-1.uIgG4.SP and WBP3248-U6T5.G25-1-uIgG4.SP to targets has not changed over time, indicating that these two antibodies bispecifics showed stability in human serum at 37 ° C for at least 14 days.

[0629] Although the disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and details can be made in them without departing from the spirit and scope of this disclosure, as disclosed in this document.

Claims (92)

1. Polypeptide complex CHARACTERIZED to comprise: a first polypeptide comprising, from the N-terminus to the C-terminus, a first heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the receptor T cells (TCR), and a second polypeptide comprising, from the N-terminus to the C-terminus, a first light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, wherein : C1 and C2 are able to form a dimer comprising at least one non-native interlink between C1 and C2, and the non-native interlink is capable of stabilizing the dimer, and the first antibody has a first antigen specificity. 2. Polypeptide complex according to claim 1, CHARACTERIZED by the fact that the non-native interchain bond is formed between a first mutated residue comprised in C1 and a second mutated residue comprised in C2. 3. Polypeptide complex according to claim 2, CHARACTERIZED by the fact that at least one of the first and second mutated residues is a cysteine residue. 4. Polypeptide complex according to any one of the preceding claims, CHARACTERIZED by the fact that the non-native interchain bond is a disulfide bond. 5. Polypeptide complex according to any one of the preceding claims, CHARACTERIZED by the fact that the first mutated residue is comprised within a C1 contact interface and / or the second mutated residue is comprised within a C2 contact interface . 6. Polypeptide complex, according to any one of the previous claims, CHARACTERIZED by the fact that at least one native cysteine residue is absent or present in C1 and / or C2. 7. Polypeptide complex, according to any of the preceding claims, CHARACTERIZED by the fact that at least one native N-glycosylation site is absent or present in C1 and / or C2. 8. Polypeptide complex according to any one of the preceding claims, CHARACTERIZED by the fact that the dimer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 non-native interchain bonds, optionally at least one of the non-native interchain bonds is a disulfide bond. 9. Polypeptide complex, according to any of the preceding claims, CHARACTERIZED by the fact that: a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama. 10. Polypeptide complex, according to any one of the preceding claims, CHARACTERIZED by the fact that: the first VH is operationally linked to C1 in a first junction domain and the first VL is operationally linked to C2 in a second junction domain. 11. Polypeptide complex according to claim 10, CHARACTERIZED by the fact that the first and / or the second junction domains comprise an appropriate length (for example, 0, 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 amino acid residues) of the C-terminal fragment of the V / C junction of the antibody and an appropriate length (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues amino acids) of the N-terminal fragment of the V / C junction of the TCR. 12. Polypeptide complex according to claim 9, CHARACTERIZED by the fact that: the modified CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35, 52-66 , 71-86 and 122-127; and / or the modified CAlfa comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 6-29, 37-67 and 86-95. 13. Polypeptide complex according to claim 9, CHARACTERIZED by the fact that the modified CBeta comprises a mutated cysteine residue that replaces an amino acid residue in a position selected from: S56C, S16C, F13C, V12C, E14C, F13C, L62C, D58C, S76C and R78C, and / or the modified CAlfa comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from: T49C, Y11C, L13C, S16C, V23C, Y44C, T46C, L51C and S62C. 14. Polypeptide complex according to claim 13, CHARACTERIZED by the fact that the modified CBeta and the modified CAlfa comprise a pair of mutated cysteine residues that replace a pair of amino acid residues selected from the group consisting of: S56C in CBeta and T49C in CAlfa, S16C in CBeta and Y11C in CAlfa, F13C in CBeta and L13C in CAlfa, S16C in CBeta and L13C in CAlfa, V12C in CBeta and S16C in CAlfa, E14C in CBeta and S16C in CAlfa, F13C in CBeta and V23C in CAlfa, L62C in CBeta and Y44C in CAlfa, D58C in CBeta and T46C in CAlfa, S76C in CBeta and T46C in CAlfa, S56C in CBeta and L51C in CAlfa, S56C in CBeta and S62C in CAlfa, and R78C in CBeta and S62C in CAlfa and in which the pair of cysteine residues are capable of forming a non-native interchain disulfide bond. 15. Polypeptide complex according to any one of claims 12 to 14, CHARACTERIZED by the fact that the native cysteine residue at position C74 of the modified CBeta is absent or present. 16. Polypeptide complex according to any one of claims 12 to 15, CHARACTERIZED by the fact that at least one native glycosylation site is absent or present in the modified CBeta and / or in the modified CAlfa. 17. Polypeptide complex according to claim 16, CHARACTERIZED by the fact that the native glycosylation site in the modified CBeta is N69 and / or the native glycosylation site (s) in the modified CAlfa is / are selected from among N34, N68, N79 and any combination thereof. 18. Polypeptide complex according to any one of claims 12 to 17, CHARACTERIZED by the fact that the modified CBeta does not have or maintain a FG loop that encompasses amino acid residues 101-117 of the native CBeta and / or a DE loop that includes amino acid residues 66-71 from native CBeta. 19. Polypeptide complex according to any one of claims 12 to 18, CHARACTERIZED by the fact that the modified CAlfa comprises SEQ ID NO: 43-48 and / or the modified CBeta comprises SEQ ID NO: 33-41. 20. Polypeptide complex according to any one of claims 10 to 19, CHARACTERIZED by the fact that C1 comprises the modified CBeta and C2 comprises the modified CAlfa; and wherein the first junction domain comprises or is SEQ ID NO: 49 or 50, and / or the second junction domain comprises or is SEQ ID NO: 51 or 52. 21. Polypeptide complex according to any one of claims 12 to 20, CHARACTERIZED by the fact that C1 comprises modified CAlfa and C2 comprises modified CBeta; and wherein the first junction domain comprises or is SEQ ID NO: 129 or 130, and / or the second junction domain comprises or is SEQ ID NO: 49 or 50. 22. Polypeptide complex according to claim 9, CHARACTERIZED by the fact that: the modified CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35, 52-66 , 71-86 and 122-127; and / or the modified CPré-Alpha comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 7-19, 26-34, 56-75 and 103-106. 23. Polypeptide complex according to claim 9, CHARACTERIZED by the fact that the modified CBeta comprises a mutated cysteine residue that replaces an amino acid residue in a position selected from: S16C, A18C, E19C, F13C, A11C, S56C and S76C and / or modified CPré-Alpha comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from S11C, A13C, I16C, S62C, T65C and Y59. 24. Polypeptide complex according to claim 23, CHARACTERIZED by the fact that the modified CBeta and the modified CPré-Alpha comprise a pair of mutated cysteine residues that replace a pair of amino acid residues selected from the group consisting of: S16C in CBeta and S11C in C Pre-Alpha, A18C in CBeta and S11C in C Pre-Alpha, E19C in CBeta and S11C in C Pre-Alpha, F13C in CBeta and A13C in C Pre-Alpha, S16C in CBeta and A13C in C Pre-Alpha, A11C in CBeta and I16C in C Pre-Alpha, S56C in CBeta and S62C in C Pre-Alpha, S56C in CBeta and T65C in C Pre-Alpha, and S76C in CBeta, and Y59C in C Pre-Alpha, and in which the pair of cysteine residues mutated cells is capable of forming a non-native disulfide bond. 25. Polypeptide complex according to any one of claims 22 to 24, CHARACTERIZED by the fact that at least one native glycosylation site is absent or present in the modified CBeta and / or the modified C-Pre-Alpha. 26. Polypeptide complex according to claim 25, CHARACTERIZED by the fact that the glycosylation site absent or present in the modified CBeta is N69 and / or the glycosylation site absent in the modified CPré-Alpha is N50. 27. Polypeptide complex according to any of claims 22 to 26, CHARACTERIZED by the fact that the modified CBeta does not have or maintain a FG loop that encompasses amino acid residues 101-107 of the native CBeta and / or a DE loop in the position encompassing amino acid residues 66-71 of native CBeta. 28. Polypeptide complex according to any one of claims 22 to 27, CHARACTERIZED by the fact that the modified CPré-Alpha comprises SEQ ID NO: 82-83, 311, 312, 313, 314, 315, 316, 317 or 318 and / or the modified CBeta comprises SEQ ID NO: 84, 33-41, 319, 320, 321, 322, 323 or 324. 29. Polypeptide complex according to any one of claims 10-11 and 22-28, CHARACTERIZED by the fact that C1 comprises the modified CBeta and C2 comprises the modified Pre-Alpha; and wherein the first splice domain comprises SEQ ID NO: 49 or 50 and / or the second splice domain comprises SEQ ID NO: 81 or 131. 30. Polypeptide complex according to any of claims 10-11 and 22-28, CHARACTERIZED by the fact that C1 comprises the modified C-Pre-Alpha and C2 comprises the modified CBeta; and wherein the first junction domain comprises SEQ ID NO: 132 or 133 and / or the second junction domain comprises SEQ ID NO: 49 or 50. 31. Polypeptide complex according to claim 9, CHARACTERIZED by the fact that: the modified CDelta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 8-26, 43-64 and 84-88; and / or the modified gamma comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 11-35 and 55-76. 32. Polypeptide complex according to claim 9, CHARACTERIZED by the fact that the modified gamma comprises a mutated cysteine residue that replaces an amino acid residue in a position selected from: S17C, E20C, F14C, T12C, M62C, Q57C and A19C and / or the modified CDelta comprises a mutated cysteine residue that replaces an amino acid residue at a position selected from: F12C, M14C, N16C, D46C, V50C, F87C and E88C. 33. Polypeptide complex according to claim 32, CHARACTERIZED by the fact that the modified CGama and the modified CDelta comprise a pair of mutated cysteine residues that replace a pair of amino acid residues selected from the group consisting of: Q57C in CGama and V50C in CDelta, A19C in CGama and E88C in CDelta, S17C in CGama and F12C in CDelta, E20C in CGama and F12C in CDelta, F14C in CGama and M14C in CDelta, T12C in CGama and N16C in CDelta, M62C in CGama and D46C in CDelta, and A19C in CGama and F87C in CDelta, and in which the pair of cysteine residues introduced is capable of forming an interchain disulfide bond. 34. Polypeptide complex according to any one of claims 31 to 33, CHARACTERIZED by the fact that at least one native glycosylation site is absent or present in the modified gamma and / or the modified CDelta. 35. Polypeptide complex according to claim 34, CHARACTERIZED by the fact that the native glycosylation site in the modified gamma is N65 and / or the native glycosylation site (s) in the modified CDelta is / are one or both of N16 and N79. 36. Polypeptide complex according to any one of claims 31 to 35, CHARACTERIZED by the fact that the modified gamma comprises SEQ ID NO: 113, or 114, 333, 334, 335, 336, 337, 338, 339 or 340, and / or the modified CDelta comprises SEQ ID NO: 115, 116, 325, 326, 327, 328, 329, 330, 331 or 332. 37. Polypeptide complex according to any of claims 10-11 and 31-36, CHARACTERIZED by the fact that C1 comprises the modified gamma and C2 comprises the modified CDelta; and wherein the first junction domain comprises SEQ ID NO: 117 or 118 and / or the second junction domain comprises SEQ ID NO: 119 or 120. 38. Polypeptide complex according to any one of claims 10-11 and 31-36, CHARACTERIZED by the fact that C1 comprises the modified CDelta and C2 comprises the modified gamma; and wherein the first junction domain comprises SEQ ID NO: 123 or 124, and / or the second junction domain comprises SEQ ID NO: 125 or 126. 39. Polypeptide complex, according to any one of the preceding claims, CHARACTERIZED by the fact that the first antigenic specificity is directed to an exogenous antigen, an endogenous antigen, a self-antigen, a neoantigen, a viral antigen or a tumor antigen. 40. Bispecific polypeptide complex, CHARACTERIZED by comprising a first antigen-binding portion associated with a second antigen-binding portion, in which: the first antigen-binding portion comprises: a first polypeptide comprising, from the N-terminal to the C-terminus - terminal, a first heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the T cell receptor (TCR), and a second polypeptide comprising, from the N-terminal end to the C- terminal, a first light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, where: C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, and the first antibody has a first antigenic specificity, a second antigen-binding portion has a second antigenic specificity that is different from the first antigenic specificity, and the first antigen-binding portion and the second antigen-binding portion are less likely to mismatch than it would be otherwise if both the first and second antigen binding portions were equivalent to natural Fab. 41. Bispecific polypeptide complex, CHARACTERIZED for comprising: a first antigen binding portion, comprising the polypeptide complex defined in any one of claims 1-39, with a first antigen specificity, associated with a second antigen binding portion with a second antigenic specificity that is different from the first antigenic specificity, and the first antigen binding portion and the second antigen binding portion are less prone to mismatch than it would otherwise be if both the first and second antigen binding portions were equivalent to natural Fab. 42. Bispecific polypeptide complex according to any one of claims 40 to 41, CHARACTERIZED by the fact that the second antigen-binding portion comprises a heavy chain variable domain and a light chain variable domain of a second antibody with the second antigenic specificity. 43. Bispecific polypeptide complex according to any one of claims 40 to 42, CHARACTERIZED by the fact that the second antigen-binding portion comprises a Fab. 44. Bispecific polypeptide complex according to either of claims 40 or 43, CHARACTERIZED by the fact that the first antigenic specificity and the second antigenic specificity are directed to two different antigens or are directed to two different epitopes on an antigen. 45. Bispecific polypeptide complex according to claim 44, CHARACTERIZED by the fact that one of the first and second antigenic specificities is directed to a specific T cell receptor molecule and / or a specific killer cell receptor molecule (NK cell) and the other targets a tumor-associated antigen. 46. Bispecific polypeptide complex according to claim 45, CHARACTERIZED by the fact that one of the first and second antigenic specificities is directed to CD3 and the other is directed to a tumor-associated antigen. 47. Bispecific polypeptide complex according to claim 46, CHARACTERIZED by the fact that one of the first and second antigenic specificities is directed to CD3 and the other is directed to CD19. 48. Bispecific polypeptide complex according to any one of claims 40 to 47, CHARACTERIZED by the fact that the first antigen-binding portion further comprises a first dimerization domain and the second antigen-binding portion further comprises a second domain of dimerization, in which the first and second dimerization domains are associated. 49. Bispecific polypeptide complex according to claim 48, CHARACTERIZED by the fact that the association is by means of a connector, a disulfide bond, a hydrogen bond, electrostatic interaction, a saline bridge or hydrophobic-hydrophilic interaction, or combination thereof. 50. Bispecific polypeptide complex according to claim 48, CHARACTERIZED by the fact that the first and / or second dimerization domains comprise at least a portion of an antibody hinge region, optionally derived from IgG1, IgG2 or IgG4. 51. Bispecific polypeptide complex according to claim 50, CHARACTERIZED by the fact that the first and / or second dimerization domains further comprise an antibody CH2 domain and / or an antibody CH3 domain. 52. Bispecific polypeptide complex according to claim 48, CHARACTERIZED by the fact that the first dimerization domain is operationally linked to the first constant region (C1) of the TCR in a third junction domain. 53. Bispecific polypeptide complex according to claim 52, CHARACTERIZED by the fact that: a) C1 comprises a modified CBeta and the third junction domain is included in SEQ ID NO: 53 or 54; b) C1 comprises a modified CAlfa and the third junction domain is included in SEQ ID NO: 134, 135, 140 or 141; c) C1 comprises a modified CPré-Alpha and the third junction domain is included in SEQ ID NO: 134, 135, 140 or 141; d) C1 comprises a modified CGama and the third junction domain is included in SEQ ID NO: 121 or 122; or e) C1 comprises a modified CDelta and the third junction domain is included in SEQ ID NO: 127 or 128. 54. Bispecific polypeptide complex according to claim 49, CHARACTERIZED by the fact that the second dimerization domain is operably linked to the heavy chain variable domain of the second antigen binding portion. 55. Bispecific polypeptide complex according to any one of claims 48 to 54, CHARACTERIZED by the fact that the first and second dimerization domains are different and are associated in a way that discourages homodimerization and / or favors heterodimerization. 56. Bispecific polypeptide complex, according to claim 55, CHARACTERIZED by the fact that the first and second dimerization domains are able to associate in heterodimers through knob-into-holes, hydrophobic interaction, electrostatic interaction, hydrophilic interaction or greater flexibility. 57. Bispecific polypeptide complex according to any of claims 40-56, CHARACTERIZED by the fact that the first antigen-binding portion comprising the first VH-containing polypeptide operably linked to a chimeric constant region, and the second polypeptide comprises VL operatively linked to C2, where the chimeric constant region and C2 comprise a pair of sequences selected from the group consisting of: SEQ ID NOs: 177/176, 179/178, 184/183, 185/183, 180/176, 181 / 178, 182/178, 184/186, 185/186, 188/187, 196/187, 190/189, 192/191, 192/193, 195/194, 198/197, 200/199, 202/201, 203/201, 203/204, 205/204, 206/204, 208/207, 208/209, 211/210, 213/212, 213/151, 214/212, 214/151, 234/233, 232 / 231, 216/215, 218/217, 220/219, 222/221, 224/223, 226/225, 227/223, 229/228, 229/230, 236/235 and 238/237. 58. Bispecific polypeptide complex according to any one of claims 40-57, CHARACTERIZED by the fact that the first antigenicity is directed to CD3 and the first polypeptide and the second polypeptide comprise a pair of sequences selected from the group consisting of: SEQ ID NOs: 2/1, 4/3, 5/1, 6/3, 7/3, 9/8, 10/8, 9/11, 10/11, 13/12, 15/14, 17/16 , 17/18, 20/19, 12/21, 65/64, 67/66, 69/68, 70/68, 70/71, 72/71, 73/71, 75/74, 75/76, 78 / 77, 86/85, 90/89, 91/92, 94/93, 96/95, 98/97, 99/95, 101/100, 101/102, 106/105, 108/107, 110/109 , 112/111, 137/136, 138/136, 137/139 and 138/139. 59. Bispecific polypeptide complex according to any of claims 40-58, CHARACTERIZED by the fact that the first antigen-binding portion is capable of binding to CD3, and the second antigen-binding portion is capable of binding CD19, and the bispecific polypeptide complex comprises a combination of four polypeptide sequences selected from the group consisting of: SEQ ID NOs: 12/22/24/23, 12/25/26/23 and 12/25/27/23. 60. Conjugate CHARACTERIZED for comprising the polypeptide complex defined in any one of claims 1-39, or the bispecific polypeptide complex defined in any one of claims 40-59, conjugated to a moiety. 61. Isolated polynucleotide CHARACTERIZED for encoding the polypeptide complex defined in any one of claims 1-39, or the bispecific polypeptide complex defined in any one of claims 40-59. 62. Isolated vector CHARACTERIZED for comprising the polynucleotide defined in claim 61. 63. Host cell CHARACTERIZED to comprise the isolated polynucleotide defined in claim 61 or the isolated vector defined in claim 62. 64. Method for expressing the polypeptide complex according to any one of claims 1-39, or the bispecific polypeptide complex according to any one of claims 40-59, characterized in that it comprises culturing the host cell defined in claim 63 under the condition in which the polypeptide complex or the bispecific polypeptide complex is expressed. 65. Method for producing the polypeptide complex according to any one of claims 1-39, or the bispecific polypeptide complex, characterized by comprising: a) introducing into a host cell: a first polynucleotide encoding a first polypeptide comprising, at the end N-terminal to C-terminal, a first heavy chain variable region (VH) of a first antibody operably linked to a first constant region (C1) of the TCR, and a second polynucleotide encoding a second polypeptide comprising, from the N- terminal to C-terminal, a first variable light chain (VL) domain of the first antibody operably linked to a second constant region (C2) of the TCR, where: C1 and C2 are capable of forming a dimer comprising at least one interchain linkage non-native between C1 and C2, and the non-native interchain bond is able to stabilize the dimer, and the first antibody has a first antigen specificity b) allowing the host cell to express the polypeptide complex. 66. The method of claim 65, further comprising: a) introducing into one host cell one or more additional polynucleotides encoding a second antigen binding portion, wherein the second antigen binding portion has a second antigenic specificity different from the first antigenic specificity, b) allowing the host cell to express the bispecific polypeptide complex. 67. The method of any one of claims 64-66, characterized in that it further comprises isolating the polypeptide complex or the bispecific polypeptide complex. 68. Composition CHARACTERIZED for comprising the polypeptide complex defined in any one of claims 1-39, or the bispecific polypeptide complex defined in any one of claims 40-59. 69. Pharmaceutical composition CHARACTERIZED for comprising the polypeptide complex defined in any one of claims 1-39, or the bispecific polypeptide complex defined in any one of claims 40-59 and a pharmaceutically acceptable carrier. 70. Method for the treatment of a condition in an individual who needs the same CHARACTERIZATION because it comprises administering to the individual a therapeutically effective amount of the polypeptide complex defined in any one of claims 1-39, or the bispecific polypeptide complex defined in any one of claims 40-59. 71. Method according to claim 70, CHARACTERIZED by the fact that the condition can be alleviated, eliminated, treated or prevented when the first antigen and the second antigen are modulated. 72. Polypeptide complex FEATURED for comprising: 1) a first antigen-binding portion comprising: a heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the T cell receptor (TCR) , and a light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, where C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first residue mutated comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, one in which a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; and 2) a second antigen-binding portion comprising: a VH of a second antibody operably linked to an antibody heavy chain CH1 domain, and a VL of the second antibody operably linked to an antibody light chain constant (CL) domain , and where the first antigen-binding portion and the second antigen-binding portion are less prone to mismatch than it would otherwise be if both the first and second antigen-binding portions were equivalent to natural Fab. 73. Polypeptide complex according to claim 72, further comprising a third antigen-binding portion comprising a VH of a third antibody operably linked to a CH1 heavy chain domain of the antibody, and a VL of the third antibody operably linked to a light chain domain CL of the antibody, wherein the CH1 of the third antigen-binding portion is operably linked to the VH of the second antigen-binding portion. 74. Polypeptide complex FEATURED for comprising: 1) a first antigen-binding portion comprising: a heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the T cell receptor (TCR) , and a light chain variable domain (VL) of the first antibody operationally linked to a second constant region (C2) of the TCR, where C1 and C2 are capable of forming a dimer comprising at least one non-native interlinked bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interlinked bond is capable of stabilizing the dimer, and wherein a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; and 2) a second antigen-binding portion comprising: a VH of a second antibody operably linked to C1 and a VL of the second antibody operably linked to C2, wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, and in which a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; 3) a third antigen-binding portion comprising: a VH of a third antibody operably linked to an antibody heavy chain CH1 domain, and a VL of a third antibody operably linked to an antibody light chain CL domain; 4) a fourth antigen-binding portion comprising: a fourth antibody VH operatively linked to an antibody heavy chain CH1 domain, and a fourth antibody VL operably linked to an antibody light chain CL domain; the polypeptide complex further comprising a first and second antibody CH2 domain and a first and second antibody CH3 domain, wherein the VH of the first antigen binding portion and the VH of the second antigen binding portion are operably linked to the first and second antibody CH3 domains, respectively, the CH1 of the third antigen binding portion and the CH1 of the fourth portion antigen binding agents are operably linked to the first and second antibody CH2 domains, respectively, and the third antigen binding portion and the fourth antigen binding portion are capable of forming a dimer. 75. Polypeptide complex FEATURED for comprising: 1) a first antigen-binding portion comprising: a heavy chain variable (VH) domain of a first antibody operably linked to a first T cell receptor (TCR) constant region (C1) , and a light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, where C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first residue mutant comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, and in which a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; and 2) a second antigen-binding portion comprising: a VH of a second antibody operably linked to C1 and a VL of the second antibody operably linked to C2, wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, and in which a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; 3) a third antigen-binding portion comprising: a VH of a third antibody operably linked to an antibody heavy chain CH1 domain, and a VL of a third antibody operably linked to an antibody light chain CL domain; 4) a fourth antigen-binding portion comprising: a VH of a fourth antibody operably linked to an antibody heavy chain CH1 domain, and a VL of the fourth antibody operably linked to an antibody light chain CL domain; the polypeptide complex further comprising a first and second antibody CH2 domain and a first and second antibody CH3 domains, wherein the C1 of the first antigen binding portion and C1 of the second antigen binding portion are operably linked to the first and second antibody CH2 domains, respectively, the VH of the third antigen binding portion and the VH of the fourth antigen binding portion are operably linked to the first and second antibody CH3 domains, respectively, and the first antigen binding portion and the second antigen-binding portion is capable of forming a dimer. 76. Polypeptide complex FEATURED for comprising: 1) a first antigen-binding portion comprising: a heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the T cell receptor (TCR) , and a light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, where C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first residue mutant comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, and in which a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; and 2) a second antigen-binding portion comprising: a VH of a second antibody operably linked to C1 and a VL of the second antibody operably linked to C2, wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, and in which a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; 3) a third antigen binding portion comprising: a VH of a third antibody operably linked to an antibody heavy chain CH1 domain, and a VL of the third antibody operably linked to an antibody light chain CL domain; 4) a fourth antigen binding portion comprising: a VH of a fourth antibody operably linked to an antibody heavy chain CH1 domain, and a VL of the fourth antibody operably linked to an antibody light chain CL domain; the polypeptide complex further comprising a first and second antibody CH2 domain and a first and second antibody CH3 domain, wherein the CH1 of the third antigen binding portion and CH1 of the fourth antigen binding portion are operably linked to the first and second antibody CH2 domains, respectively, the C1 of the first antigen-binding portion is operably linked to the VH of the first antigen-binding portion, the C1 of the second antigen-binding portion is operably linked to the VH of the second antigen-binding portion antigen, and the third antigen-binding portion and the fourth antigen-binding portion are capable of forming a dimer. 77. Polypeptide complex FEATURED for understanding: 1) a first antigen-binding portion comprising: a heavy chain variable domain (VH) of a first antibody operably linked to a first constant region (C1) of the cell receptor T (TCR), and a light chain variable domain (VL) of the first antibody operably linked to a second constant region (C2) of the TCR, where C1 and C2 are capable of forming a dimer comprising at least one non-native interlinked bond between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interlinked bond is capable of stabilizing the dimer, and wherein a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; and 2) a second antigen-binding portion comprising: a VH of a second antibody operably linked to C1 and a VL of the second antibody operably linked to C2, wherein C1 and C2 are capable of forming a dimer comprising at least one non-native interchain link between a first mutated residue comprised in C1 and a second mutated residue comprised in C2, and the non-native interchain bond is able to stabilize the dimer, and in which a) C1 comprises a modified CBeta and C2 comprises a modified CAlfa; b) C1 comprises a modified CAlfa and C2 comprises a modified CBeta; c) C1 comprises a modified CBeta and C2 comprises a modified C Pre-Alpha; d) C1 comprises a modified CPré-Alpha and C2 comprises a modified CBeta; e) C1 comprises a modified CGama and C2 comprises a modified CDelta; or f) C1 comprises a modified CDelta and C2 comprises a modified CGama; 3) a third antigen-binding portion comprising: a VH of a third antibody operably linked to an antibody heavy chain CH1 domain, and a VL of a third antibody operably linked to an antibody light chain CL domain; 4) a fourth antigen-binding portion comprising: a fourth antibody VH operatively linked to an antibody heavy chain CH1 domain, and a fourth antibody VL operably linked to an antibody light chain CL domain; the polypeptide complex further comprising a first and second antibody CH2 domain and, optionally, a first and second antibody CH3 domain, wherein the C1 of the first antigen binding portion and C1 of the second antigen binding portion are operably linked to the first and second antibody CH2 domains, respectively, the CH1 of the third antigen-binding portion is operably linked to the VH of the first antigen-binding portion, the CH1 of the fourth antigen-binding portion is operably linked to the VH of the second portion antigen-binding, and the first antigen-binding portion and the second antigen-binding portion are capable of forming a dimer. 78. Polypeptide complex according to any one of claims 72 to 77, CHARACTERIZED by the fact that: the modified CBeta comprises a mutated cysteine residue within a contact interface selected from the group consisting of: amino acid residues 9-35 , 52-66, 71-86 and 122-127; and / or the modified CAlfa comprises a mutated cysteine residue within a contact interface selected from a group consisting of: amino acid residues 6-29, 37-67 and 86-95. 79. Polypeptide complex according to claim 78, CHARACTERIZED by the fact that the modified CBeta and the modified CAlfa comprise a pair of mutated cysteine residues that replace a pair of amino acid residues selected from the group consisting of: S56C in CBeta and T49C in CAlfa, S16C in CBeta and Y11C in CAlfa, F13C in CBeta and L13C in CAlfa, S16C in CBeta and L13C in CAlfa, V12C in CBeta and S16C in CAlfa, E14C in CBeta and S16C in CAlfa, F13C in CBeta and V23C in CAlfa, L62C in CBeta and Y44C in CAlfa, D58C in CBeta and T46C in CAlfa, S76C in CBeta and T46C in CAlfa, S56C in CBeta and L51C in CAlfa, S56C in CBeta and S62C in CAlfa, and R78C in CBeta and S62C in CAlfa, and where the pair of cysteine residues are capable of forming a non-native interchain disulfide bond. 80. Polypeptide complex according to claim 79, CHARACTERIZED by the fact that the modified CBeta comprises S56C and the modified CAlfa comprises T49C. 81. Polypeptide complex according to any one of claims 72-80, CHARACTERIZED by the fact that the native cysteine residue at position C74 of the modified CBeta is absent. 82. Polypeptide complex according to any one of claims 72-81, CHARACTERIZED by the fact that at least one native glycosylation site is absent in the modified CBeta and / or in the modified CAlfa. 83. Polypeptide complex according to claim 82, CHARACTERIZED by the fact that the native glycosylation site in the modified CBeta is N69 and / or the native glycosylation site (s) in the modified CAlfa is / are selected from among N34, N68, N79 and any combination thereof. 84. Isolated polynucleotide CHARACTERIZED for encoding the polypeptide complex defined in any one of claims 72-83. 85. Isolated vector CHARACTERIZED for comprising the polynucleotide defined in claim 84. 86. Host cell CHARACTERIZED to comprise the isolated polynucleotide defined in claim 84 or the isolated vector defined in claim 85. 87. Composition CHARACTERIZED for comprising the polypeptide complex defined in any one of claims 72-83. 88. Pharmaceutical composition CHARACTERIZED for comprising the polypeptide complex defined in any one of claims 72-83 and a pharmaceutically acceptable carrier. 89. Method for treating a condition in an individual who needs the same CHARACTERIZATION because it comprises administering to the individual a therapeutically effective amount of the polypeptide complex defined in any one of claims 72-83. 90. Polypeptide complex according to any of claims 9 to 21 and 72 to 83, CHARACTERIZED by the fact that at least one native Ser residue in the modified CAlfa is mutated to reduce O-glycosylation. 91. Polypeptide complex according to any one of claims 40 to 56, CHARACTERIZED by the fact that C1 or C2 comprises a modified CAlfa and in which at least one native Ser residue in the modified CAlfa is mutated to reduce O-glycosylation. 92. Polypeptide complex according to any one of claims 90-91, CHARACTERIZED by the fact that the mutated amino acid residue is selected from S19, S36, S41, S91 and S94.